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(CANCER RESEARCH 38, 1584-1588, June 1978] 0008-5472/78/0038-OOOOS02.00 Differences in Total Mitochondrial Proteins and Proteins Synthesized by Mitochondria from Rat Liver and Morris Hepatomas 9618A, 5123C, and 5123tc1 Carol C. Irwin,2 Leonard I. Malkin, and Harold P. Morris Department ot Biochemistry, Wayne State University School ol Medicine, Detroit, Howard University School of Medicine, Washington, D. C. 20001 ¡H.P. M.] ABSTRACT Mitochondria were isolated from a slow-growing (9618A) and two intermediate-to-fast-growing (5123C, 5123tc) Mor ris hepatomas and host livers. The mitochondria! proteins were solubilized and fractionated on sodium dodecyl sulfate:polyacrylamide slab gels. One Coomassie bluestained band was absent or reduced in amount in all tumors relative to host livers. In addition, a major mito chondria! enzyme present in normal liver, carbamyl phos phate synthetase, was missing or greatly reduced in the slow-growing, highly differentiated hepatoma 9618A, a tumor that is considered to be similar to normal liver in many biochemical and morphological respects. Incuba tion of mitochondria with [35S]methionine and a suitable amino acid incorporation system resulted in labeling of specific mitochondria! proteins. Autoradiography of the slab gels disclosed four prominently labeled fractions and a number of minor fractions. Preparations from hepatoma 5123tc demonstrated two labeled bands that were absent or greatly reduced in host liver. Host liver preparations displayed a minor band that was absent or greatly re duced in hepatoma 5123C. However, no single change in labeling pattern was common to all three tumors, sug gesting the absence of a causal relationship between carcinogenesis and mutations in mitochondria! DMA. INTRODUCTION The DNA of mammalian mitochondria has a molecular weight of approximately 107 and codes for at least 2 rRNA molecules, several tRNA molecules, and several proteins, all of which are unique to mitochondria (13). Only 5 to 10% of total mitochondrial proteins are synthesized by mitochondrial ribosomes. Evidence suggests that these proteins are coded for by mitochondrial DNA. These proteins are closely associated with the inner membrane of mitochon dria, and they are insoluble except in detergents such as sodium dodecyl sulfate, so that their identification has been difficult. Only 8 proteins have been positively identified as belonging to specific mitochondrial membrane:enzyme complexes (13). Malignant transformation of cells is accompanied, to a ' Supported by the National Cancer Institute (CA 15539), the National Science Foundation (BMS73-02031), the Research Corporation (BH512), and in part by USPHS Grant CA 10729. 2 Fellow of the National Cancer Institute (CA 05109). Present address: Eunice Kennedy Shriver Center for Mental Retardation, Waltham, Mass. 02154. 3To whom requests for reprints should be addressed. Received August 26. 1977; accepted March 7, 1978. 1584 Michigan 48201 [C. C. I., L. I. M.¡,and Department oÃ-Biochemistry, greater or lesser degree, by altered enzyme activities and ultrastructure of mitochondria (16). These alterations could arise from mutations in either nuclear or mitochondrial DNA or both. Chang ef a/. (2) have demonstrated modified electrophoretic properties of 1 inner membrane protein in mitochondria from several Morris hepatomas. We have examined mitochondria from 3 hepatomas different from those studied by Chang ef a/. (2), and we have found 1 Coomassie blue-stained band absent or greatly reduced in all 3 tumors. We have established that this is not a mitochondrially synthesized protein and probably not coded for by mitochondrial DNA. Hepatoma 9618A, a very-slow-grow ing minimal-deviation tumor, lacks a major Coomassie blue-stained band that is present in host liver and the other hepatomas that have been studied. Based on the work of Clarke (3), this is almost certainly carbamyl phosphate syn thetase, an enzyme that is present in large amounts in the livers of ureotelic organisms. This protein is also not coded for by mitochondrial DNA. We have used a sensitive autoradiographic method for detecting proteins synthesized specifically by mitochondria in an in vitro amino acid incorporation system, and we have observed a larger number of labeled proteins than has previously been reported by most workers with gel slicing techniques. This observation agrees with recent reports on the labeling of mitochondrial proteins by yeast (4) and by animal tissue culture cells (6). These proteins presumably are coded for by mitochondrial DNA. Hepatoma 5123tc, a fast-growing tumor, yielded 2 labeled bands that were absent or much less conspicuous in all the other tumors and host livers. The other fast-growing hepatoma, 5123C, and a slow-growing hepatoma, 9618A, did not differ from their host livers in major respects. MATERIALS AND METHODS Animals and Hepatomas. Male tumor-bearing Buffalo rats were obtained from the colony of Dr. Harold P. Morris and kept under standard conditions. All tumors were car ried i.m. by bilateral inoculation into thigh muscle. Hepa toma 9618A (generation 10) was a highly differentiated, slow-growing tumor (transplantation time, approximately 12 months) that has been reported to have a normal chro mosome number and karyotype (12). Hepatomas 5123C (generation 120) and 5123tc (generation 150) were fast growing (transplantation time, 1 to 2 months) with a lesser degree of differentiation. Previous generations of 5123C and 5123tc have been reported to have abnormal chromo some numbers but minimal chromosome changes (12). CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1978 American Association for Cancer Research. Mitochondria! Protein Synthesis in Rat Liver and Hepatoma Hepatomas from 1 to 3 animals were pooled for each experiment. Isolation and Purification of Mitochondria. All solutions were prepared 1 to 2 days before each experiment and were autoclaved. Glassware was also sterilized. Procedures throughout the isolation of mitochondria were performed at 0-4°. Rats were killed by decapitation, and host livers were quickly removed and placed into approximately 3 volumes of 10 mM Bicine:4 2 mM EDTA:0.3 M sucrose buffer (pH 7.5). The liver was cut into small pieces with scissors and washed with fresh buffer 3 times by settling and dé cantation. Buffer was then added to 2 volumes of liver wet weight and homogenized with a motor-driven, Teflon:glass homogenizer with a clearance of about 0.25 mm. The motor was driven at 500 rpm and only 2 up-and-down strokes were used. The homogenate was diluted with another 6 volumes of buffer and centrifuged at 1400 x g for 6 min in 50-ml polycarbonate tubes, with caps, in the No. 276 swinging bucket rotor of an EC refrigerated centrifuge. The supernatant solution was separated by décantation from the light brown loose layer of the pellet, then passed through 2 layers of cheesecloth, and recentrifuged as above. The supernatant solution from the second centrifugation was placed in clean 50-ml polycarbonate tubes and centrifuged at 6000 x g for 10 min in a Sorvall refrigerated centrifuge. The supernatant solution was decanted off of the loose brown layer, and the pellet was partially resuspended with a "cold finger" (sterile 15-ml test tube filled with ice). Resuspension was completed by adding a small amount of buffer and drawing up the suspension into a cotton-plugged, sterile 10-ml blow-out pipet, followed by gravity out-flow, a total of 2 to 3 times. The suspension was diluted to a final volume of one-half of the original volume and recentrifuged for 10 min at 6000 x g. The pellet was resuspended into one-fourth of the original volume of buffer prior to the final centrifugation, after which all the supernatant solution was discarded, including any loose material. The final pellet was resuspended to a final concen tration of approximately 25 mg mitochondrial protein per ml. In the case of hepatomas, tumors were removed from the thigh muscle of the rats after they had attained a diameter of approximately 2 to 3 cm. Tumors of this size were seldom necrotic. Nontumorous tissue or, rarely, necrotic tissue was carefully trimmed away prior to isolation of mitochondria, as described for host liver. The only difference in procedure was the increase in homogenization from 2 strokes to 10. The characterization of these mitochondria preparations has been reported previously (11). Amino Acid Incorporation. The complete incubation sys tem was based on that of Malkin (11) and contained 10 mM Bicine, 10 mM K2HPO4, 2 mM EDTA, 0.154 M KCI, 10 mM MgCI2, 1 mM ATP, 5 mM phosphoenolpyruvate, 20 /xg pyruvate kinase, 19 amino acids (minus u-methionine) (2 /xg each), 0.5 mg cycloheximide, 1 to 2 mg mitochondrial protein, and 0.14to 0.25 mCi L-[35S]methionine (Amersham/ Searle Corp., Arlington Heights, III.). Specific activity of the L-[35S]methionine was 5 to 6 Ci/mole. Final volume was 1 ml and the final pH was 7.4. Chemicals were purchased * The abbreviation used is: Bicine, W,/V-bis(2-hydroxyethyl)glycine. from Sigma Chemical Co., St. Louis, Mo. Stock solutions of Bicine, K,HPO4, EDTA, KCI, and MgCI, were freshly prepared and autoclaved 1 to 2 days before use. Other stock solutions were prepared with sterile water. The basic incorporation mix, before addition of mitochondria and L[35S]methionine, was filtered immediately before use with a Millipore bacteriological filter. After incubation for 1 hr at 37°in glass Sorvall centrifuge tubes, the incorporation mixtures were placed on ice and diluted with 5 ml wash buffer [0.25 M sucrose, 0.01 M Tris (pH 6.7), 0.025 M KCI, and 0.005 M MgCL] containing cold L-methionine (1 mg/ml). Mitochondria were pelleted by centrifugation at 12,000 x g for 10 min in the SS34 rotor in a Sorvall refrigerated centrifuge. Pellets were washed twice and then dissolved in 0.5 ml of a dissociating medium, modified from that of Douglas and Butow (4), containing 2% sodium dodecyl sulfate, 0.002 M EDTA, 0.2% (v/v) 2mercaptoethanol, 0.05 M Tris (pH 6.8), and 10% (v/v) glycerol. The pellets were heated for 2 min at 90°and then either stored overnight at room temperature or used imme diately for electrophoresis. Overnight incubation did not alter either stained or labeled banding patterns. Protein Assay. Protein was estimated by the method of Lowry ef al. (10). In the case of mitochondrial protein dissolved in electrophoresis medium, correction was made for the artifactual contribution of 2-mercaptoethanol. Bacterial Counting. Control tubes that contained incor poration mix, mitochondria, but no isotopes were incu bated in parallel with expérimentais. One-tenth-ml aliquots were plated on blood agar and incubated for 72 hr at 37°, and the number of colonies was determined. Slab Gel Electrophoresis and Autoradiography. The general procedures for electrophoresis and autoradiography were those of Douglas and Butow (4). Approximately 50 /xg of mitochondrial protein per well were layered on the 15-cm slab gels described by Studier (14). The discontin uous buffer system of Laemmli (8) was used, except that 0.002 M EDTA was included in the stacking and resolving gels, and 0.5% polyacrylamide (BDH Laboratories, GallardSchlesinger Chemical Mfg. Corp., Carle Place, N. Y.) was included in the resolving gel to ensure even drying of the gel for autoradiography (4). The resolving gel (7.5 to 12% linear acrylamide gradient) was prepared 1 to 2 days before hand, and the 5% stacking gel was prepared immediately prior to electrophoresis. Up to 13 samples of mitochondrial or marker protein could be subjected to electrophoresis at 1 time. Electrophoresis was performed at 30 ma constant current until the bromphenol blue tracking dye reached 1 cm from the bottom of the slab (about 5 hr). The gels were dried either immediately after electropho resis, or after staining, on Whatman No. 3MM paper over a steaming water bath under reduced pressure. For staining, gels were soaked in 10% trichloroacetic acid at 70°for 20 min, stained in methanohacetic tained 0.05% (w/v) Coomassie acid:water (5:1:5) that con blue at 37°overnight, and destained in 7.5% acetic acid that contained 5% methanol. If stained gels were to be dried, they were soaked for 2 hr in 1% glycerol. Dried gels were exposed from 3 weeks to 2 months to Kodak RP X-omat medical X-ray film that was then developed in Kodak liquid X-ray developer for 5 min at 22°. JUNE 1978 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1978 American Association for Cancer Research. 1585 C. C. Irwin et al. Marker Proteins. Molecular weight estimations on gels the host liver. were made with marker proteins: /3-lactoglobulin (M.W. Because of the variation in tumor growth, mitochondrial 18,000); chymotrypsinogen A (M.W. 26,000); ovalbumin (M. proteins from the various hepatomas had to be subjected to W. 43,000); and serum albumin (M.W. 67,000). ß-Lactoglob- electrophoresis separately, resulting in slight variations in ulin migrates somewhat anomalously in our gel system, at length of run and gel composition. In addition, there was an apparent molecular weight of 15,000. All marker pro an apparent variation in the extent of labeling (note the teins were purchased from Sigma Chemical Co., St. Louis, largar number of bands detected in mitochondria from Mo., except for the chymotrypsinogen A (Worthington Bio 9618A and 5123C). We believe that these experimental chemical Corp., Freehold, N. J.). variations account for the small apparent differences seen among host livers, although the possibility of actual varia tions in individual host livers cannot be completely ex RESULTS cluded. We have consistently observed, however, that la Coomassie Blue-stained Gels: Total Proteins. The band beled proteins from several normal livers yield identical ing patterns for all 3 hepatomas and their host livers are patterns when run on the same gel. shown in Fig. 1. When allowance is made for slight differ Bacterial contamination was negligible, as shown by the ences due to separate gel runs, the banding patterns are results of blood agar plating. The mitochondrial incubation identical among host livers. In general, mitochondrial pro mixture contained less than 200 bacteria/ml. teins from each hepatoma bear great similarity to those of their host livers. There are several exceptions to this, DISCUSSION however. One prominent difference between all 3 hepato The incorporation of [35S]methionine into mitochondrial mas and their respective host livers is the absence or proteins of both normal liver and hepatomas was carried reduction of a polypeptide with an approximate molecular out in the presence of cycloheximide, an inhibitor of cytoweight of 36,000 in tumor mitochondria (dashed arrow). Other differences can be detected between tumor and host plasmic but not mitochondrial protein synthesis (11). There fore cytoplasmic protein synthesis does not contribute to liver mitochondria, but these are minor and are not com labeling of the proteins in the system used (Fig. 2) although mon to all of the hepatomas that are studied. The most striking difference is the absence in hepatoma minor bands might not have been detected in these experi 9168A of the high-molecular-weight polypeptide that ap ments. In addition, the level of bacterial contamination in pears to be the most prominent band in host liver (so//'d these experiments is much lower than that needed (30,000 arrow). Although we have not definitively identified this bacteria/ml) to show significant bacterial amino acid incor polypeptide by biochemical means, the position of the band poration (11). and the absence or reduction of the band in non-liver tissue Autoradiographic studies of mitochondrially synthesized such as brain, kidney, and heart, as well as fetal liver proteins indicate that there are 2 additional (or more highly labeled) bands in Morris hepatoma 5123tc compared with (unpublished results), indicate that this polypeptide is prob ably the protein with the molecular weight of 165,000, host liver mitochondria. Except for the absence or reduc carbamyl phosphate synthetase, observed by Clarke (3) to tion in label of a minor band in mitochondria from hepa occur in liver of ureotelic organisms. This protein makes up toma 5123C, no other differences between tumor and host 15 to 20% of the total mitochondrial protein in normal liver livers were detectable. Apparently no single alteration in mitochondrial translation products is common to all hepa (3). Mitochondrially Synthesized Proteins. Incubation of iso tomas; therefore the role of mitochondrial DNA in tumorilated mitochondria with [35S]methionine of very high spe genesis remains uncertain. It has been reported that mito cific activity, along with an amino acid incorporation sys chondria do preferentially accumulate certain carcinogenic tem, resulted in sufficient labeling of mitochondrially syn hydrocarbons (5) and also that mitochondrial DNA may be thesized proteins to allow autoradiography. The results of 3 particularly sensitive to mutagenesis since it lacks tightly such experiments are shown in Fig. 2. When mitochondrial bound proteins (15). Certain carcinogenic hydrocarbons do proteins from each hepatoma are compared with those of in fact preferentially interact with mitochondrial DNA as its host liver, hepatoma 5123tc is the only one of the 3 opposed to nuclear DNA (17). It would be of interest to tumors that differs greatly from the host. Culture cells from establish whether the band pattern of hepatoma 5123tc is which tumor 5123tc was derived came from generation 20 due to an alteration of mitochondrial DNA itself or to a of tumor line 5123C, and the lines have not been mixed in subsequent step in the translation process. It is possible subsequent generations. that the changes seen are the indirect effect of cytoplasmic There are 4 major labeled bands (marked with solid changes that are known to affect in large degree such arrows) in all the gels. In 5123tc appear 2 labeled bands mitochondrial functions as the proper assembly of enzyme (dashed arrows) that are absent or greatly reduced in host complexes (e.g., cytochrome oxidase) that originate both from nuclear and mitochondrial gene products (13). liver. These bands have molecular weights of approximately 15,000 and 31,000 (based on molecular weight standards The autoradiographic method used in this study allows that were run on the same gel). Hepatoma 5123C also much better resolution than has previously been obtained differs from its host control by the absence or reduction in with the widely used gel slicing technique (e.g., Refs. 1 and 9). Our results substantially agree with another radiolabeling of a minor band of approximately 60,000 (dotted arrow). By contrast, the minimal-deviation hepatoma 9618A graphic study by Jeffreys and Craig (6), who distinguished apparently has no labeled proteins that differ from those of 7 to 11 labeled major bands and a number of minor bands 1586 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1978 American Association for Cancer Research. Mitochondria! Protein Synthesis in Rat Liver and Hepatoma from mitochondria of animal culture cells. Douglas and Butow (4) have resolved 21 labeled bands from yeast mito chondria that were labeled under conditions that excluded cytoplasmic protein synthesis. Whether all 21 proteins in yeast, or in the animal mitochondria that have been studied, are unique gene products as opposed to aggregates and/or cleavage products remains to be seen. Kuzela ef al. (7) conducted a study in which mitochondria from rat liver and Zajdela hepatoma were labeled in vivo with [14C]leucine in the presence of cycloheximide. The distribution of label in sodium dodecyl sulfate:polyacrylamide gels was determined by the gel slicing technique and was the same in both cases. The study was limited, however, by the low resolution of gel slicing and the low level of radioactivity in the labeled bands. While it is possible that in fact no actual differences existed between the hepatoma in question and normal liver, as we found in 1 of our hepatomas, the difference that we detected with the autoradiographic procedure would have gone unno ticed by the method of Kuzela ef a/. (7). When total mitochondrial proteins by means of Coomassie blue staining rather than specific mitochondrial transla tion products were considered, we found 1 difference that was common to all the hepatomas studied, namely, the absence or reduction of a band with a molecular weight of approximately 36,000. This agrees with the report of Chang ef a/. (2), who analyzed mitochondrial membrane proteins from another series of hepatomas of varying degrees of differentiation. However, the molecular weight of their pro tein was not determined, and therefore the identity of the protein with ours cannot be established. The polypeptide that we observed to be missing is apparently not a mitochondrially synthesized protein, inasmuch as it did not coincide with any of the bands in the autoradiographs. The absence in hepatoma 9168A of the major band near the top of the gel seen in normal liver is somewhat surpris ing because this tumor is highly differentiated and very slow growing, a type of tumor that differs little in many respects from normal tissue. The work of Clarke (3) sug gests that this band corresponds to carbamyl phosphate synthetase, a polypeptide with a molecular weight of 165,000. Clarke found that this band is reduced in fetal liver (3), and we have established the same phenomenon with the band in question (unpublished data). The enzyme pat terns in many transformed cells conform to fetal patterns, but the observation of such a relationship in a highly differ JUNE entiated tumor, especially when such a pattern is not seen in faster-growing, less differentiated tumors, is striking. In summary, the absence of a common difference in mitochondrially synthesized proteins (comparing 3 hepato mas to host liver) would seen to argue against a causal relationship between carcinogen induction of hepatomas and specific mutations in mitochondrial DNA. REFERENCES 1. Burke, J. P., and Beatile, D. S. Products of Rat Liver Mitochondrial Protein Synthesis: Electrophoretic Analysis of the Number and Size of These Proteins and Their Solubility in Chloroform:Methanol. Arch. Biochem. Biophys., 764: 1-11, 1974. 2. Chang, L. O., Schnaitman, C. A., and Morris, H. P. Comparison of the Mitochondrial Membrane Proteins in Rat Liver and Hepatomas. Cancer Res.,3i: 108-113, 1971. 3. Clarke, S. 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Lederman, M., and Attardi. G. Expression of the Mitochondrial Genome in HeLa Cells XVI. Electrophoretic Properties of the Products of In Vivo and In Vitro Mitochondrial Protein Synthesis. J. Mol. Biol., 78: 275-283, 1973. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with the Polin Phenol Reagent. J. Biol. Chem., 793: 265275, 1951. 11. Malkin, L. I. Amino Acid Incorporation by Isolated Rat Liver Mitochondria during Liver Regeneration. Proc. Nati. Acad. Sei. U. S.. 67: 1695-1702, 1970. 12. Nowell, P. C., Morris, H. P., and Potter, V. R. Chromosomes of "Minimal Deviation" Hepatomas and Some Other Transplantable Rat Tumors. Cancer Res.. 27: 1565-1579, 1967. 13. Schatz, G., and Mason, T. L. The Biosynthesis of Mitochondrial Proteins. Ann. Rev. Biochem., 43: 51-87, 1974. 14. Studier. F. W. Analysis of Bacteriophage T7 Early RNAs and Proteins. J. Mol. Biol., 79: 237-248, 1973. 15. Swift, H., Rabinowitz. M., and Getz. G. Cytochemical Studies on Mito chondrial Nucleic Acids. In: E. C. Slater, J. M. Tager. S. Papa, and A. Quagliariello (eds.). Biochemical Aspects of the Biogenesis of Mito chondria, p. 3. Bari, Italy: Adriatica Editrice, 1968. 16. Wallach, D. F. H. Cellular Membranes and Tumor Behavior: A New Hypothesis. Proc. Nati. Acad. Sei. U. S., 67: 866-874, 1968. 17. Wunderlich, V., Schutt, M., Bottger, M., and Graffi, A. Preferential Alkylation of Mitochondrial Deoxynucleic Acid by A/-Methyl-/V-nitrosourea. Biochem. J., 778: 99-109, 1970. 1978 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1978 American Association for Cancer Research. 1587 C. C. Irwin et al. 9618A T L —165 1-165 -67 -43 x * -26 -15 -15 I 2 Fig. 1. Comparison of electrophoretic properties of total Coomassie blue-stained mitochondrial proteins from hepatomas (7")and host livers (L). The cath ode is at the fop. Fig. 2. Comparison of autoradiographs of 35S-labeled mitochondrially synthesized proteins from hepatomas (7) and host livers (i.). The cathode is at the top. 1588 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1978 American Association for Cancer Research. Differences in Total Mitochondrial Proteins and Proteins Synthesized by Mitochondria from Rat Liver and Morris Hepatomas 9618A, 5123C, and 5123tc Carol C. Irwin, Leonard I. Malkin and Harold P. Morris Cancer Res 1978;38:1584-1588. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/38/6/1584 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1978 American Association for Cancer Research.