Characterization and Subunit Analysis of Ferritin Isolated from Normal and Malignant Human Liver'

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[CANCER RESEARCH 35, 1505-1509, June 1975] Characterization and Subunit Analysis of Ferritin Isolated from Normal and Malignant Human Liver'

Elliot Alpert Departmentsof Medicine, Harvard Medical School and Gastrointestinal Unit, MassachusettsGeneralHospital, Boston, Massachusetts02114

SUMMARY nant cells in tissue culture and rat hepatoma migrated more rapidly electrophoretically than normal. We have confirmed Ferritin was purified from normal, fetal, and malignant and extended these observations by showing that ferritin liver tissue. Ferritin purified from hepatoma tissue migrated isolated from human liver carcinoma and fetal liver differed slightly faster than normal human liver ferritin in polyacryl from normal liver ferritin and contained isoferritin variants, amide gel eiectrophoresis. Hepatoma and fetal liver ferritin not present in normal liver, which we have called carcino contained an acidic component in gel and liquid isoelectric fetal isoferritins (I). Therefore, in an attempt to character focusing not found in normal liver ferritin. We have called it ize the molecular basis for the additional carcinofetal acidic a carcinofetai isoferritin. The subunit compositions of isoferritin, we have studied the subunit structure of ferritin ferritins purified from human liver cell carcinoma and isolated from normal and malignant liver tissue. normal liver were then compared. Both ferritins consisted of a subunit species with an identical molecular weight of approximately 18,500. A single subunit of similar molecular MATERIALS AND METhODS weight was also demonstrable after dissociation of 8 M urea and by gel filtration in urea. Two subunits were demonstra Fresh tissues were obtained postmortem from the Dc ble in normal liver ferritin by means of acrylamide electro partment of Pathology, Massachusetts General Hospital, phoresis in 8 M urea in acid pH. The same two subunits were Boston, Mass., within 12 hr after death. Fetal liver was ob also demonstrable in ferritin isolated from human liver cell tamed from therapeutic abortions, performed for medical carcinoma. However, a third subunit, intermediate in legal reasons during early pregnancy. The tissues were charge between the two normal liver subunits, was demon stored at â€”20Â°until the ferritin was extracted, and the strable in different amounts in ferritins from two hepato extracted ferritin was kept at 4Â°.Ferritin was isolated from mas. Ferritins from normal and malignant livers were these tissues by the method of Drysdale and Munro (5), immunologically indistinguishable. The tumor-specific except that carboxymethylcellulose chromatography was acidic isoferritin was isolated and antisera were prepared. omitted to avoid possible selective loss of isoferritins of The isolated acidic isoferritin was found to be immunoiogi differing charge. The tissues were homogenized and sub caiiy identical to normal liver isoferritins. It is concluded jected to heat and acid precipitation, ammonium sulfate that the multiple isoferritins of the human liver ferritin precipitation, and final purification by Sephadex G-200 consist of two subunits, which are identical in molecular column chromatography (5). Sephadex was purchased weight but which differ in net charge. Ferritin, isolated from from Pharmacia Fine Chemicals Inc., Piscataway, N. J. two human liver carcinoma tissues, was composed of the The purity of each preparation was confirmed by poiy same two subunits and a third unique subunit. Different acrylamide gel electrophoresis, a commonly accepted amounts of these subunits may account for the several presumptive evidence for purity of ferritin. In each case, normal isoferritins and a unique tumor-specific acid isofer only bands that stained for both protein and iron were seen ritin found in hepatoma. in positions where ferritin is known to migrate. Slab electrophoresis was performed using a system purchased from Ortec Inc., Oak Ridge, Tenn. A vertical INTRODUCTION quartz cell with internal dimensions of 9.5 cm x 9 cm x 4 mm was filled with 4.5% poiyacrylamide Eastman Organic An increasingbodyof evidenceindicatesthatthe iron Chemicals, Rochester, N. Y. that was water layered to form storage protein, ferritin, is composed of multiple molecular a straight edge and then allowed to polymerize. After forms or isoferritins. These isoferritins have been separated removing the water layer, the gel slab was overlaid with 8% by isoelectric focusing or ion-exchange chromatography (4, polyacrylamide gel, containing a Teflon well former, and 21, 22). Of particular interest was the observation by allowed to polymerize for 20 mm. After removal of the Richter and Lee ( 17, 18) that ferritin isolated from malig Teflon well former, the wells were rinsed with distilled water and blotted dry. Ferritin samples, purified according to the identical protocol and containing 50 to 100 @zgof protein, 1 This work was supported by NIH Grant CA-12389 and American Cancer Society Grant IM-l4B. were each thoroughly premixed with an equal volume of Received October 11, 1974; accepted February 21, 1975. 50% sucrose in 0.0375 M Tris-sulfate, pH 9.0, and placed in

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. E. A!pert the well. Electrophoresis was performed vertically, with the SDS to enhance dissociation into its subunits (20). They cathode uppermost, in 0.065 MTris-borate buffer, pH 9.0, at were then subjected to SDS-polyacrylamide gel electra 4Â° and a constant voltage from an Ortec 4l00 pulsed phoresis (12) (Chart 2). The gels were stained for protein, constant power supply. The pulse rate was increased at each gel was analyzed by UV densitometry, and migration 5-mm intervals, from 75 to a maximum of 300 pulses/sec at indexes were calculated and compared to standards. The 15 mm. The total running time was approximately 45 mm. major subunit of each ferritin migrated between myoglobin The gel slab was stained for protein using Ponceau S or and chymotrypsinogen and was calculated by densitometry Coomassie blue and counterstained for iron with potassium to weigh 18,500 daltons. In addition, there were 2 smaller ferrocyanide. SDS2-polyacrylamide electrophoresis was fragments with molecular weights of approximately 9,000 performed according to the method of Neville (12). Molecu and 11,000 noted in lower concentration. The same pattern lar weights were estimated from precise densitometric was reproduced in mixing experiments, where malignant measurements of protein bands compared to standards. and normal liver ferritin were mixed and reanalyzed by SDS Acid urea electrophoresis was performed in a thin gel slab of eiectrophoresis showing the major subunit to be indistin 4.5% polyacrylamide in 6 M urea and 20 mr@iglycine-HCI guishable in size by this technique. buffer at pH 3.0. Purified ferritin preparations were first Ferritins purified from normal, fetal, and malignant liver dialyzed against 6 M urea at pH 3.0 and then placed in the tissue were subjected to preparative isoelectric focusing in a wells. The buffer trays contained acid glycine-HCI buffer, sucrose density gradient. Quantitative estimation of the 0.2 M in 6 M urea, and electrophoresis was performed at a apoferritin by electroimmunodiffusion revealed 6 or 7 peaks constant current of 30 ma for about 3 hr. in preparation. When the fractions were plotted by the pH Preparative isoelectric focusing was performed in a at which they focused as the ordinate (Chart 3), it was 110-mi sucrose density gradient containing 4% ampholytes evident that the ferritin from malignant liver had its major (LKB Productor, Sweden), pH 5 to 7. After 3 to 5 days of peaks focusing at a pH more acidic than normal. Hepatoma electrolysis, at an initial constant power setting of 2.5 watts ferritin had isoferritin peaks common to both normal and until equilibrium was achieved, 1.0-mi fractions were col fetal liver ferritin. lected from the bottom of the column, and the pH gradient However, the single most acidic isoferritin fraction was measured with a microelectrode. Apoferritin was identified eluted at pH 5.05, a pH where no normal ferritin was and quantitated by electroimmunodiffusion (9) using mono detectable on subsequent focusing runs (Chart 3). This specific rabbit anti-human ferritin. Analytic gel dcc fraction was shown by analytical gel focusing to be free of trofocusing was performed according to the method of the isoferritins characteristic of normal liver tissue. This Righetti and Drysdale (19) in 4.5% polyacrylamide. The gels isolated hepatoma specific isoferritin was mixed with equal were stained for protein with Coomassie blue, immuno volumes of Freund's complete adjuvant and injected into the precipitated in situ with anti-liver ferritin antisera (4, 16), or stained for iron by potassium ferrocyanide. Acid urea gel filtration was performed in Sephadex G-75 equilibrated with 8 M urea and 0.02 Mglycine-HC1 buffer, pH 3.0. The + column was calibrated by means of dextran blue, chymo trypsinogen, and myoglobin standards Pharmacia, Uppsala, Sweden. RESULTS

Ferritin was purified from primary liver cell carcinoma and normal adult liver tissue and compared by polyacryl amide gel electrophoresis in a vertical slab at alkaline pH (Chart I). All samples were purified according to the same protocol outlined in â€œMaterialsand Methodsâ€•and were dialyzed against the same barbital buffer, pH 8.6, to equalize the pH and ionic strength of the samples prior to electrophoresis. All the bands were stained for both protein and iron, identifying them as ferritin. Small amounts of dimer and oligomeric ferritins were also visualized but no contaminating non-iron-containing proteins were seen, demonstrating the purity of the preparation. The mono meric form of ferritin isolated from liver cell carcinoma H .N consistently migrated slightly ahead of ferritin isolated from normal liver. Chart 1. Polyacrylamide gel electrophoresis at pH 8.6. Ferritin isolated Ferritins isolated from liver cell carcinoma, fetal liver, from hepatoma(H) andnormal adult liver (N) subjectedto electrophoresis and normal liver were incubated at 60Â°for 1 hr in 0.1% after dialysis againstthe samebarbital buffer, pH 8.6, to equalizepH and ionic strength in the samples. All bands stained for iron and protein with

2 The abbreviation used is SDS, sodium dodecyl sulfate. the major band representingthe ferritin monomer.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. Norma! and Hepatoma Ferritin Subunits, Isoferritins lower-molecular-weight fragments previously identified also .4- @.- I@. appeared in the identical place in most of the peaks tested, suggesting further degradation of the major subunit into 2 smaller fragments. In order to determine whether the major subunit of @ @li! ferritin showed variation in net charge, experiments were -MY performed in 8 M urea in acid pH. The major subunit of -CT hepatoma ferritin was isolated from its partially degraded oligomeric forms by gel filtration on Sephadex G-75 equilibrated in 8 Murea, pH 3.0 (Chart 4). The column was calibrated with dextran blue, chymotrypsinogen, and myo -OA globin. The incompletely denatured higher-molecular -BSA weights fragments eluted in the void volume. A single symmetrical peak was seen in the inclusion volume, eluting just ahead of myoglobin, consistent with a single subunit of 18,500 daltons in urea, and identical to that determined by SDS gel eiectrophoresis. Aliquots of ferritin isolated from 2 hepatoma livers and

H N FStd.

Chart 2. SDS-polyacrylamidegel clectrophoresisof ferritins isolated from hepatoma(H), normal liver (N), and fetal liver (F). Tracking dyewas marked with India ink to allow precisemeasurementof R@,.Standards included myoglobin (MY), chymotrypsinogen(CT), ovalbumin (OA), and bovine serum albumin (BSA). The major ferritin subunit (arrow) weighed 18,500 daltons.

footpads of 2 rabbits. The rabbits were given boosters at 4 weeks and bled 2 weeks later. The antisera were tested by Ouchterlony gel diffusion against the acidic isoferritin used as the immunizing agent, purified hepatoma ferritin, and purified normal liver ferritin. A line of complete identity was observed with the anti-acid isoferritin antisera against all 3 ferritin preparations. The same result was obtained using antisera made against purified normal ferritin. No spurring was noted. Cross-absorption studies were also

performed. Anti-acid isoferritin antisera were absorbed with I' normal liver ferritin, and anti-normal liver ferritin was absorbed by hepatoma ferritin. After this cross-absorption, both antisera did not react with any of the ferritin prepara tions. These data suggest that the tumor-specific acid isoferritin contains no exposed antigenic determinant that could be differentiated from normal liver ferritin by the immunological means used. The major peaks, as shown in the brackets in Chart 3, as well as the more acidic and basic fractions on either side, were pooled from normal, hepatoma, and fetal ferritin after separation by preparative isoelectric focusing. The isolated

isoferritins were dialyzed back to pH 7.4 and reanalyzed by IIII gel isofocusing. The isoferritins redistributed according to 4.9 5.1 5.3 5.5 5.7 the characteristic p1 of each isoferritin, as previously demonstrated (4, 7). Aliquots were dialyzed against 0.1% pH SDS; heated to 60Â°for 60 mm, and the 3 fractions of each Chart 3. Preparative isoelectric focusing of ferritin isolated from normal, fetal, and hepatoma ferritin were then compared by normal, fetal, and hepatomalivers in a sucrosedensitygradient using4% SDS gel electrophoresis. The major subunit, from all ampholines,pH 5 to 7. Fractions, I .0 ml, were eluted and the pH was isoelectric focusing fractions in each preparation, had an determined. The ferritin protein concentration was measuredimmuno identical molecular weight of 18,500 as measured by chemically by electroimmunodiffusion. The main peaks (brackets) were densitometry and calculation of mobility. In addition, the 2 pooled and comparedto more acidic and basicfractions.

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E. A!peri

Sephodu G-75, 8M Ursa demonstrated that human livers tumors and fetal liver ferritin contain a unique acid isoferritin band, which we Void volume Chymo-trypsinoqsn Myoglobin have called a carcinofetal isoferritin (17). Similar acidic ferritins have been shown to occur in human pancreatic and breast tumors (1 1) and in HeLa cells and early placental extracts (6). Since these multiple forms are not likely to be 80 due to a single uniform subunit population, we investigated the subunit structure of ferritins isolated from hepatoma 90 and normal liver. The molecular weight of horse spleen ferritin subunits @ II IIIIII I 0 10 20 30 40 50 60 70 80 90 $00 110 was found to be 18,500 by SDS (2). Niitsu et a!. (13) have demonstrated subunit heterogeneity in horse spleen ferritin FRACTION NUMBER which we have confirmed (unpublished observations). How Chart 4. Urea gel filtration in SephadexG-75equilibratedwith 8 Murea ever, we found only a single major subunit with a molecular in 0.02 M glycine-HCI, pH 3.0. The column was calibrated with dextran weight of 18,500 in normal human liver ferritin by SDS gel blue, chymotrypsinogen, and myoglobin. eiectrophoresis. We were unable to demonstrate the exist ence of any major subunit differences in molecular weight in several normal livers were then compared by acid urea gel hepatoma or normal liver ferritin by SDS gel electrophore electrophoresis. The samples were first dialyzed against 8 M sis or by urea gel filtration. However, trace amounts of 2 urea in 0.02 M glycine-HCI buffer, pH 3.0, and subjected to lower-molecular-weight fragments were seen in several electrophoresis in acrylamide in the same acid urea condi preparations of human ferritin, with molecular weights of tions. Two bands (A and B) were consistently noted in approximately 9,000 and I 1,000 by SDS electrophoresis. ferritin isolated from normal liver, both migrating slightly These appear to correspond to the lower-molecular-weight slower than cytochrome c. The same 2 bands were noted in fragments found by Niitsu et a!. (I 3) in horse spleen and the hepatoma ferritins although with very different intensi human ferritin. The acidic isoferritin had a molecular ties. Furthermore, there was an additional ferritin subunit weight identical to the basic bands. No additional bands band (C) intermediate between the 2 normal bands, particu were detected after careful densitometry measurements, or larly evident in the 1st hepatoma (H1) (Chart 5). by coelectrophoresis. Gel filtration in urea also demon The same preparations were also subjected to urea strated only a single symmetrical peak in the calibrated electrophoresis in 7, 11, and 13.5% polyacrylamide. Electro included volume, indicating a molecular weight of about phoresis in different polyacryiamide gel concentrations had 18,500. Thus, the major subunit size found in urea was no effect on the relative migration of the major subunit essentially the same as that previously demonstrated by bands (A, B, and C). All additional bands disappeared after SDS gel electrophoresis. treatment with dithiothreitoi and were also excluded from Normal liver ferritin consisted of 2 distinctly different the higher gel concentrations. The hepatoma ferritin (Hi) electrophoretic forms of subunits (A and B) when subjected that had higher concentration of the unique tumor specific to acid urea electrophoresis. The relationship between the 2 intermediate band, and less of the normal band B, also subunit bands did not change when analyzed in 4 different showed a higher concentration of the tumor-specific acid acrylamide concentrations, confirming that the difference isoferritin on gel electrofocusing (1). was due to charge differences only. All slower bands were removed from normal ferritin by either treatment with DISCUSSION dithiothreitol or $-mercaptoethanol, as noted by Niitsu and Listowsky (14), or by the higher concentrations of acrylam Ferritin is an intracellular iron storage protein that ide. This indicated that the additional components excluded consists of a hollow protein shell (apoferritin) in which at higher gel concentrations were aggregates probably inorganic iron may be sequestered (7). It has been thought bound by disulfide bonds. Our demonstration of 2 distinct that ferritin consisted of 20 or 24 subunits (2, 8), which were normal liver ferritin subunits of similar molecular weight, thought to be identical (8). However, multiple molecular but differing in charge, may explain the microheterogeneity forms of ferritin, or isoferritins, have recently been demon strated in horse spleen, rat liver, and human liver by @@@ electrofocusing (1 , 4, 15, 2 1, 22) and ion-exchange chroma A A â€¢.A tography (3, 4, 21, 22). These forms have been shown to be @ stable molecular forms unchanged by the pH alterations on B . B *@J@ reanalysis (1, 7). We have previously shown that ferritin isolated from a human liver tumor has more rapid electro @ phoretic mobility than normal liver ferritin (1) and appears N Hi:';@ . . H.2. to be similar to the more rapidly migrating â€œabnormalâ€• Chart 5. Urea gel electrophoresis in 4.5% polyacrylamide equilibrated in ferritins found in malignant cell cultures (17), the livers of 6 M urea in 0.02 M glycine-HCI buffer, pH 3.0. Ferritins isolated from tumor-bearing animals (10, 18). We have found similar normal liver (N) and hepatoma livers (H, and H2) were dialyzed and changes in ferritin isolated from early fetal livers and have equilibrated in the same 0.02 M glycine-HCI buffer, pH 3.0, and 8 M urea.

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of ferritin previously reported (3, 4, 6, 13, 16, 21, 22). The Isoferritin in Placenta and HeLa Cells. Cancer Res., 34. 3352-3354, multiple isoferritins, separated by isoelectric focusing or 1974. ion-exchange chromatography, could consist of ferritin 7. Granick, S. Ferritin: Its Properties and Significance for Iron Metabo shells containing different proportions of the 2 sUbunits. lism. Chem. Rev., 38: 379-403, 1946. A 3rd unique subunit (Band C) with an intermediate net 8. Hofmann, T., and Harrison, P. M. The Structure of Apoferritin: Degradation into and Molecular Weight of Subunits. J. Mol. Biol., 6: charge has been demonstrated in the 2 hepatomas tested. 256-257,1963. The hepatoma and normal ferritin were immunologically 9. Laurell, C. B. Quantitative Estimation of Proteinsby Electrophoresis indistinguishable. Nevertheless, the subunits may contain in Agarose Gel Containing Antibodies. Anal. Biochem., 15: 45-52, unique antigenic determinants hidden within the shell and 1966. need to be further characterized. The presence of an 10. Linder, M., Munro, H. N., and Morris, H. P. Rat Ferritin Isoproteins additional tumor-specific subunit in the ferritin shell could and Their Responseto Iron Administration in a Series of Hepatic be responsible for the presence of the additional acidic Tumors and in Normal and Regenerating Liver. Cancer Res., 30: isoferritin found in human hepatoma ferritin (1). This acid 2231â€”2239,1970. isoferritin is present in fetal liver in early gestation, 11. Marcus, D. M., and Zinberg, N. Isolation of Ferritin from Human disappears in late gestation, and reappears in cancer tissue Mammary and Pancreatic Carcinomas by Means of Antibody Im munoadsorbents.Arch. Biochem. Biophys., 162:493-501. 1974. (1). This suggests that the acid isoferritin and apparently 12. Neville, D. M. Molecular Weight Determination of Protein Dodecyl unique subunit may represent another carcinofetai altera Sulfate Complexes by Gel Electrophoresis in a Discontinuous Buffer tion of protein synthesis in tumors. System. J. Biol. Chem., 246: 6328-6334, 1971. 13. Niitsu, Y., Ishitani, Y., and Listowsky, I. Subunit Heterogeneity in Ferritin. Biochem. Biophys. Res. Commun., 55. 1134-1 140, 1973. ACKNOWLEDGMENTS 14. Niitsu, Y., and Listowsky, I. Mechanisms for the Formation of Ferritin Oligomers. Biochemistry, 12: 4690â€”4695,1973. We wish to gratefully acknowledgetheexcellenttechnicalassistanceof 15. Powell, L., Alpert, E., Drysdale, J. W., and Isselbacher, K. J. Organ Ruth L. Coston,whosepatienceandperseverancemadethis studypossible. Specific Forms of Ferritin in Normal Human Tissues and Cirrhosis We also wish to thank Dr. James W. Drysdale for reviewing this and Iron Storage Disease. Gastroenterology, 64: 889, 1973. manuscript and for his continued interest and collaboration. 16. Powell, L. W., Alpert, E., Drysdale, J. W., and Isselbacher,K. J. Abnormality in Tissue Ferritin in Idiopathic hemochromatosis. Na ture, 250: 333â€”335,1974. REFERENCES 17. Richter, G. W. Comparison of Ferritins from Neoplastic and Non neoplastic Human Cells. Nature, 207: 616â€”618,1965. I. Alpert, E., Coston, R. L., and Drysdale, J. W. Carcino-Fetal Human 18. Richter, G. W., and Lee, J. C. I. C. A Study ofTwo Types of Ferritin Liver Ferritins. Nature, 242: 194-196, 1973. From Rat Hepatoma. Cancer Res., 30: 880â€”888,1970. 2. Crighton, R. R., and Bryce, C. F. Molecular Weight Estimation of 19. Righetti, P. G., and Drysdale, J. W. Isoelectric Focusing in Polyacryl Apoferritin Subunits. Federation European Biochem. Soc. Letters, 6: amide Gels. Biochim. Biophys. Acta, 236: 17â€”28,1971. 121-124,1970. 20. Smith-Johannsen, H., and Drysdale, J. W. Reversible Dissociation of 3. Drysdale,J. W. Micro Heterogeneityin Ferritin Molecules.Biochim. Ferritin and Its Subunits in Vitro. Biochim. Biophys. Acta, 194: Biophys. Acta, 207: 256-258, 1970. 43-49, 1969. 4. Drysdale, J. W. Heterogeneityin Tissue Ferritins Displayed by Gel 21. Urushizaki, I., Ishitani, K., and Niitsu, Y. Microheterogeneity of Rat Electrofocusing. Biochem. J., 141: 627â€”632,1974. Liver Ferritin: Comparison of Electrofocusing and Chromatographic 5. Drysdale, J. W., and Munro, H. M. Small Scale Isolation of Ferritin Fractions. Biochim. Biophys. Acta. 328: 95-1 10, 1973. for the Assay of the Incorporation of â€œC-labeledAminoAcids. 22. Urushizaki, I., Niitsu, Y., Ishitani, Y., Matsuda, M., and FuKuda, Biochem. J., 95: 851â€”858,1965. M. Microheterogeneity of Horse Spleen Ferritin and Apoferritin. 6. Drysdale, J. W., and Singer, R. M. Carcinoma Fetal Human Biochim. Biophys.Acta. 243: 187â€”192,1971.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. Characterization and Subunit Analysis of Ferritin Isolated from Normal and Malignant Human Liver

Elliot Alpert

Cancer Res 1975;35:1505-1509.

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