Biochemical Properties of Neoplastic Cell Mitochondria 1,2

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1 Biochemical Properties of Neoplastic Cell Mitochondria 1,2 Michael T. White,3.4 D. V. Arya,5 and K. K. Tewari 4.6 SUMMARY-Mitochondria from monolayer cultures of Novikoff hepatomacells were studiedfor their biochemical and structural organization and compared with mitochondria from solid Novikoff hepatoma and normal liver. Cultured cell and tumor mitochondria were similar in both ultrastructure and biochemical properties. Both differed distinctly from liver mitochondria, in that they had drastically reduced activities of several flavoprotein enzymes associated with their outer and Inner membranes and altered morphology of their matrix as evident from electron micrographs. The alterations we observed In the Novlkoff hepatoma mitochondria were not the result of varied growth conditions but were Inherent In these neoplastic cells. Mitochondria from another neoplastic cell line, hamster melanoma CCl 49, grown in vitro were compared to mitochondria from cultures of a non-neoplastic cell line, hamster embryo Nil 2. Melanoma cell mitochondria showed reductions In outer and inner membrane f1avoprotelns similar to those observed in Novlkoff hepatoma mitochondria. There were significant reductions In the activities of NADH oxidase, NADH-cytochrome c reductase, and adenylate kinase. In addition, monoamine oxidase was absent.-j Natl Cancer Inst 53: , WE RECENTLY reported that mitochondria from Novikoff hepatoma had drastic alterations in their biochemical properties and structural organization (1). For example, flavoprotein enzymes associated with the outer and inner membranes had significantly lower activities in mitochondria of tumors compared to those in liver. Electron microscopy revealed a disorganized internal structure in tumor mitochondria, and these organelles were heterogeneous in size and density. These studies were done on mitochondria isolated from serially transplanted, solid tumors; it can be argued that the aberrations observed might be the result of physiologic growth conditions, i.e., poor blood supply and low O 2 tensions. To eliminate this possibility, we have now extended our studies on Novikoff hepatoma mitochondria to the more precisely defined and controlled conditions of in vitro, monolayer cultures of these hepatoma cells. The biochemical and structural characteristics of mitochondria from these cells are similar, if not identical, to solid tumor mitochondria. We concluded that the mitochondrial aberrations observed in the Novikoff hepatoma are an inherent property of these neoplastic cells and not related to growth conditions. Further investigating the role of the mitochondrion in neoplasia, we extended our studies to normal and neoplastic cells in monolayer culture. Mitochondria from the Syrian hamster melanoma cell line (CCL 49) were compared to those of a normal Syrian hamsterembryo cell line (Nil 2). Cytochrome oxidase and malate dehydrogenase activities were similar in melanoma and embryo cell mitochondria, but adenylate kinase activity was reduced 65% in melanoma mitochondria. A striking observation was the complete absence of monoamine oxidase activity in melanoma mitochondria and the reduction by 70 80% of several electron-transport flavoprotein enzymes. Thus the pattern of biochemical abnormalities in melanoma mitochondria closely parallels that observed in the Novikoff hepatoma. MATERIALS AND METHODS Tumors and cell culture.-sprague-dawley female rats (Holtzman Laboratories, Madison, Wis.) were fed a standard stock diet (Wayne Lab Blox, Allied Mills, Chicago, Ill.) and used to serially maintain Novikoff hepatomas as described previously (1). A cell line derived from the Novikoff hepatoma was obtained from Dr. L. Bradshaw (California State University, Fullerton, Calif.). Cells from this culture were passed in a ratio of I :2, divided into 25 fractions of 5 ml each, and stored at - 70 C. Fresh cells were periodically thawed and cultured from this frozen stock, so that cells were maintained for :l> passages. Cells were grown in monolayer culture in Eagle's minimum essential medium containing 2 msr L-glutamine, 10% fetal bovine serum (Grand Island Biological Co., Menlo Park, Calif.), and antibiotics (100 U penicillin/ml and 100 /lg streptomycin/rnl). Cells were cultured in either 32-ounce prescription bottles (Sani-glass, Brockway Glass Co., Covina, Calif.) or disposable cell-production roller bottles (Belko Glass Co., Vineland, N.J.) in a 5% CO 2-air, humidified atmosphere at 37 C. CCL 49 was obtained from the American Type Culture Collection. It was established by Moore et al. (2) from a malignant melanoma that arose spontaneously in an aged Syrian hamster and was carried by subcutaneous implantations for 41 passages by Fortner et al. (3). For use as normal hamster cells, a clone derived by McAllister et al. (4) from Nil 2 was obtained from Dr. S. Rasheed (University of Southern California, School of Medicine, Los Angeles, Calif.). Both of these cell lines were cultured in McCoy's 5a medium containing 10% fetal bovine serum and antibiotics. 1 Received March 4, 1974; accepted April 18, Supported in part by Public Health Service NIH ~ostdoctoral' Fellowship 2 FO CA5151G--02 from the National Cancer Institute to MTW. 3 Present address: Cancer Research Laboratory, University of California, Berkeley, Calif Department of Molecular Biology and Biochemistry, University of California, Irvine, Calif Estelle Doheny Eye Foundation Laboratory, Los Angeles, Calif We gratefully acknowledge the technical assistance of Ms. Marcia Rice and 'Ms. Juta Kiethe in preparation of the electron micrographs. JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 53, NO.2, AUGUST

2 554 WHITE, ARYA, AND TEWARI Preparation of mitochondria.-cells were harvested with a rubber policeman and washed 3 times in phosphate-buffered saline, ph 7.0 (3.2 rnn NaHP04 l2 H 20; 1.5 rna KH 2P04 ; 137 mu NaCl; 3 mx KCI; 0.9 rmr CaCI 2 ; and 0.5 mm MgC1 2 6 H 20). Cell pellets were resuspended in cold sucrose medium (SM) (0.25M sucrose; 2 mx ethylenediaminetetraacetic acid; and 50 mm Tris-HCI, ph 7.4) containing 1% bovine serum albumin (BSA). They were disrupted by strokes at 5 C in a stainless-steel dounce fitted with a polished steel pestle (clearance = 0.05 mm). Three volumes of SM containing 1% BSA were added to the homogenate, the solution was centrifuged at 500Xg for 10 minutes, and the crude mitochondrial pellet was obtained by centrifugation at 8700 Xg for 15 minutes. The white fluffy layer of the pellet was discarded. The mitochondria were then washed in 30 ml cold SM-l % BSA, recentrifuged at 8700 Xg for 15 minutes, further purified by centrifugation in linear sucrose or discontinuous BSA gradients, and assayed for enzyme activity. Mitochondria from solid tumors and liver were obtained as previously described (1). Enzyme assays.-we assayed cytochrome oxidase by spectrophotometrically measuring the oxidation of reduced cytochrome c as described by Smith (5). Malate dehydrogenase was tested by the method of Ochoa (6), adenylate kinase by the technique of Schnaitman and Greenawalt (7), and NADH-cytochrome c (cyt c) reductase by the method of Sottocasa et al. (8). Rotenone sensitivity was measured by preincubation of the assay mixture in 5 J.Lg rotenone before addition of substrate. We tested NADH oxidase by measuring the disappearance of NADH spectrophotometrically (9). Succinate dehydrogenase activity was determined, as described by Bonner (10), in briefly sonicated mitochondria. Succinate oxidase was measured with a vibrating oxygen electrode (1). To measure monoamine oxidase activity, 2 substrates were used: Oxidation of the first substrate, 14C tryptamine (47.3 mcijmmole; New England Nuclear, Boston, Mass.), was assayed by the method of Wurtman and Axelrod (11), and oxidation of the second substrate, 14C-serotonin (26.7 mcijmmole; New England Nuclear), by the procedure of McCaman et al. (12). All enzymes were assayed for maximum activity and, whenever noted, mitochondria were disrupted by the addition of 0.2 mg of the nonionic detergent Lubrol WX (ICI America, Charlotte, N.C.)jmg protein before the enzymes were tested. Except when specified, all enzymatic activities were assayed with the use of mitochondria isolated in SM-I% BSA and purified on sucrose or BSA gradients. For any given enzyme, liver, tumor, and cultured hepatoma cell mitochondria were concurrenly tested, as were melanoma and embryo cell mitochondria. All enzymatic activities reported were the averages of at least 2 separate experiments. Host and normal liver mitochondria were extensively compared and did not differ appreciably in regard to either specific activity of mitochondrial enzymes, number of mitochondriaj mg mitochondrial protein, density in sucrose and BSA gradients, and ultrastructural morphology [(1) and unpublished results]. Thus in studying the activities of various mitochondrial specific enzymes, we used host liver for comparison with solid hepatoma and cultured hepatoma cells. Protein was analyzed by the method of Lowry et al. (13) with BSA as a standard. Electron microscopy.-fresh tissues or cell pellets were fixed in a small volume of 4% glutaraldehyde in 0.1M phosphate buffer (ph 7.1) for 2 hours at 25 0 C. Glutaraldehyde was removed by 3 successive washes in phosphate buffer. The samples were postfixed in 2% OS04 in phosphate buffer (ph 7.2) plus 0.05% CaC1 2 for 2 hours at 25 0 C and then dehydrated in an acetone series. A low-viscosity epoxy resin (14) was used as the embedding medium. Sections were cut with a diamond knife on an LKB microtome and stained with 2% uranyl acetate and lead citrate (15). Electron micrographs were taken on the Zeiss EM 59 microscope operating at 50 kv. RESULTS Novikoff Hepatoma Density-gradient profiles.-the equilibrium densities of liver, solid Novikoff hepatoma, and cultured tumor cell mitochondria were determined by centrifugation in linear lM sucrose gradients. Fractions were collected and assayed for protein, refractive index, and the activities of cytochrome oxidase and monoamine oxidase (text-fig. I). Cultured tumor cell mitochondria had a density of gjml, slightly lighter than the density of gjml for the solid tumor mitochondria. Both mitochondria were significantly lighter than liver mitochondria, which had a density of gjml (text-fig. I, B-D). Changes in the densities of solid tumor and cultured tumor cell mitochondria were also seen when mitochondria were banded in discontinuous BSA gradients (35, 29, 26, 23, and 10%) and assayed for monoamine oxidase activity (text-fig. 2). Liver mitochondria formed a single, dense band in the 35% region of the gradient (text-fig. 2); monoamine oxidase activity was not found in any other region of the gradient. By the same criterion, solid tumor mitochondria were heterogeneous. A third of the mitochondria banded in the 35% BSA region, another third in the 29% BSA region, and the rest in the 26% BSA region. Cultured tumor cell mitochondria were homogeneous and were in a single region of the gradient (23% BSA). Thus, by the criteria of density in sucrose and BSA gradients, Novikoff hepatoma mitochondria from both solid tumor and cultured tumor cells were distinctly altered compared to liver mitochondria. Electron microscopy.-mitochondrial ultrastructure was examined in electron micrographs of thin sections of liver, solid tumor, and cultuerd tumor cells (figs. la-c). Liver mitochondria had a characteristic ultrastructure with a well-defined outer membrane, numerous cristae, and a dense-staining matrix (fig. Ia), The mitochondria of solid tumor and cultured tumor cells were similar but distinctly different from liver mitochondria in respect to the internal matrix.

3 BIOCHEMICAL PROPERTIES OF NEOPLASTIC CELL MITOCHONDRIA 555 A. B. NOVIKC F HEPATOMA CELLCULTURE NOVIKC F HEPATOMA CELLCULTURE 18 IrIII. +DENSITY ;/l1li n -< 'DENSln... 4, R :x i '" i 0 X 0 X i! e l/l lit '" /'; ~ <: oj :::; < -< =< 100 -< E ::> <, 3 co 0 10 ii Q. e c ii' lit D. LIVER NOVIKOFF HEPATOMA, Ii i s» Z 3: '" ~ +D HSITy...m z 0 Z '"!! 0 0 X» 300 III ~ 500'" /'; /'; ~ <: :I :::; -< B 5 35T B T FRACTION NUMBER TEXT-FIGURE I.-Sucrose gradient profiles of normal liver, solid Novikoff hepatoma, and cultured hepatoma cell mitochondria isolated in SM-I% BSA. Gradients and mitochondria were prepared as described in "Materials and Methods." Fractions were assayed for refractive index, protein (el, and cytochrome oxidase or monoamine oxidase activity (.). Bo th tumor and cultured tumor cell mitochondria had a dilute, lightly stained matrix (figs. 1b, c). Enzymatic Activities Isolated mitochondria from host liver, solid tumor, and cultured tumor cells were assayed for a number of mitochondrial specific enzymes. Some enzymes were chosen for diverse mitochondrial location and others for their role in electron transport (table 1). The activity of cytochrome oxidase,' on the inner mitochondrial membrane (16), was similar in host liver, solid tumor, and cultured tumor cell mitochondria. The activity of malate dehydrogenase in the matrix (16) was also approximately the same in these mitochondria. Adenylate kinase in the space between the inner and outer membrane (16) was reduced 60% in cultured tumor cell mitochondria and 30% in solid tumor mitochondria when compared to those of host liver. Rotenone-insensitive NADH-cyt c reductase in the outer membrane (8) was reduced 60% in both solid tumor and cultured tumor cell mitochondria compared to those in host liver. Monoamine oxidase, also restricted to the outer membrane (8), was 60% lower than that in host liver in both solid tumor and cultured tumor cell mitochondria. The same level of reduction was found for the oxidation of both serotonin and tryptamine. When the electrontransport flavoproteins were considered, NADH-cyt c reductase had a fourfold decrease in activity in mitochondria of both solid tumor and cultured tumor cells compared to that in host liver. Similarly, NADH oxidase was reduced eightfold, whereas succinate dehydrogenase and succinate oxidase showed fourfold to fivefold decreases in solid tumor and cultured tumor cell mitochondria compared to those in host liver. Solid tumor and cultured tumor cell mitochondria had nearly the same activities for the various enzymes studied (table I). All mitochondria isolated by our methods exhibited respiratory control; it is therefore unlikely that the changes in enzymatic activities we observed were artifacts of isolation. Melanoma and Embryo Cells The densities of melanoma and embryo cell mitochondria in linear sucrose gradients were both g/m!. Also, densities did not differ in discontinuous BSA gradients, where mitochondria from both cell types banded at the 23% BSA region of the gradient. Enzymes were assayed in mitochondra isolated from both cell types (table 2). Melanoma and embryo cell mitochondria had similar cytochrome oxidase and malate dehydrogenase activities. However, adenylate kinase activity was reduced by 60% in the melanoma

4 556 WHITE, ARYA, AND TEWARI >-~ t:,~ ~o t- h. ~ C' 160 w- (f) c: «0 'E - " o x- g 100 w"8 ~a «Cl> :::E '" 00 Z E o c: :::E ~ 20 TEXT-FIGURE 2.-Banding of normal liver, solid Novikoff hepatoma, and cultured hepatoma cell mitochondria in discontinuous BSA gradients. Gradients and mitochondria were prepared as described in "Materials and Methods." Mitochondria, banding in the various regions of the gradients, were collected, washed in SM, and assayed for monoamine oxidase activity with l~-tryptamineas substrate, mitochondria. Furthermore, monoamine oxidase activity was not detected in melanoma mitochondria. Embryo mitochondria had 72 U of activity with 14C-tryptamine as substrate and 220 U with 14C serotonin as substrate, but no melanoma activity was detected with either of these substrates. Also, rotenonetnsensitive NADH-cyt c reductase was reduced by 60% in the melanoma mitochondria, and the electrontransport flavoproteins -NADH-cyt c reductase and NADH oxidase-were reduced 70 and 80%, respectively. The reductions in enzymatic activities of melanoma cells parallel those seen in the Novikoff hepatoma. DISCUSSION LIVER QI NOVIKOFF HEPATOMA o NOVIKOFF HEPATOMA CELL CULTURE PELLET 35-29" % 26-23" % PERCENT BSA The density of cultured tumor cell mitochondria in sucrose gradients was g/ml compared to g/ml of host liver mitochondria. Density of tumor mitochondria was g/ml. Thus both solid tumor and cultured tumor cell mitochondria had reduced densities compared to those of host liver mitochondria. The mitochondria from all these sources were not fragmented during isolation, since there was good coincidence between the banding of mitochondrial protein and the mitochondrial marker enzymes cytochrome oxidase and monoamine oxidase. Density differences were also seen in the banding patterns of mitochondria in discontinuous BSA gradients. Solid tumor mitochondria were heterogeneous in density and banded in 3 regions of the gradient. Cultured tumor cell mitochondria were homogeneous and banded at a single, lighter region of the gradient. Mitochondria from solid tumor and cultured tumor cells not only differed from one another but also were distinctly lighter than liver mitochondria. The differences in density between solid tumor and cultured tumor cell mitochondria can be understood in light of the fact that the solid hepatoma is a heterogeneous mass of cells, whereas the cultured tumor cells represent a clone of one or a few cell types from the tumor. Nevertheless, the reduction in mitochondrial density was retained in both solid tumor and cultured tumor cells, which indicates that changes in the densities of mitochondria are an inherent property of the Novikoff hepatoma. Other similarities between solid tumor and cultured tumor cell mitochondria and differences of both from liver mitochondria were seen in electron micrographs of these tissues. Solid tumor and cultured tumor cell mitochondria had dilute, lightly stained matrices. In contrast, liver mitochondria under similar experimental conditions had a condensed, densely stained matrix. No other ultrastructural differences were evident. Changes in the matrix of these tissues and cells seen in thin section correlate with the dilute, disorganized matrix seen in electron micrographs of isolated Novikoff hepatoma mitochondria (1). Further, isolated hepatoma mitochondria appeared to have intact outer membranes, and TABLE I.-Enzymatic activities of sucrose gradient-purified mitochondria isolated in media containing 1% BSA Specific activity (nmoles product formed/min/mg protein)*, Novikoff hepatoma cells Novikoff hepatoma Host liver ~tochromeoxidaset c alate dehydrogenaset Adenylate kinase t Monoamine oxidase (HC-tryptamine) Monoamine oxidase (HC-serotonin) N AD H-cyt c reductase Rotenone-insensitive NADH-cyt c reductase NADH oxidase] Succinate dehydrogenase; Succinate oxidase ~. 513± ±180 83±11 66±3 461±32 124± ±4 35±2 42±4 9±2 575± ± ±24 76±5 427±38 93±8 72±3 34±3 94±8 4±1 611± ± ±33 171±1O 1255± ±37 246± ±24 435±65 33±5 Mean:!:SD of 4 determinations in 2 or 3 separate experiments. Number of mitochondria/mg mitochondrial protein: liver=4.73xlo'; tumor=4.92x10'; and hepatoma cells=4.51xlo'. tpretreated with Lubrol WX.

5 BIOCHEMICAL PROPERTIES OF NEOPLASTIC CELL MITOCHONDRIA 557 TABLE 2.-Enzymatic activities of sucrose gradient-purified mitochondria isolated in media containing 1% BSA Specific activity (nmoles product formed/rnin/mg protein)* Cytochrome oxidase] - - Malate dehydrogenaset Adenylate kinaset ---- Monoamine oxidase (14C-tryptamine) Monoamine oxidase (14C-serotonin) NADH-cyt o reductase Rotenone-insensitive NADH-cyt c reductase NADH oxidase] Melanoma cells 403± ±103 71±10 <0.01 < ±30 136± 12 73±6 Hamster embryo cells 613± ± ±31 72±8 220± ± ±34 417±46 *Mean±BDof 4 determinations in 2 or 3 separate experiments. [Pretreated with Lubrol WX. no correlation was attempted between membrane ultrastructure and reduced enzyme activity (1). Similarly, in electron micrographs of isolated, cultured, tumor cell mitochondria, intact outer membranes and dilute, disorganized matrices were evident (unpublished results). It is still difficult to correlate specific ultrastructural alterations with specific biochemical changes in hepatoma or cultured hepatoma cell mitochondria. Isolation and characterization by electrophoresis of mitochondrial membrane proteins would be useful in an attempt to correlate the observed biochemical and ultrasturctural abnormalities. When the activities of a number of important mitochondrial specific enzymes were compared, cytochrome oxidase and malate dehydrogenase had similar levels in host liver, solid tumor, and cultured tumor cell mitochondria. Similar results have been reported in other hepatomas (17, 18). The activity of adenylate kinase in cultured tumor cell mitochondria was 83 U compared to 171 U for the solid tumor mitochondria and 222 U for host liver mitochondria. A reduction in adenylate kinase activity has been reported in other fast-growing hepatomas (18-20). This enzyme has been shown to exist in several isozymic forms, and the change from one form to another or the reduction in total activity could decrease the ability of -mitochondria to regulate the levels of adenine nucleotide pools necessary for the regulation of a wide range of metabolic pathways (19). Flavoprotein enzymes associated with the mitochondrial electron-transport chain were reduced to the same degree in both solid tumor and cultured tumor cell mitochondria compared to those of host liver. NADH-cyt c reductase, NADH oxidase, succinate dehydrogenase, and succinate oxidase were reduced fourfold to eightfold in both solid tumor and cultured tumor cell mitochondria. The reduction of these enzymes in the Novikoff hepatoma could reflect either a reduction in all the mitochondria or lowered activity in some mitochondria while others contain normal levels of activity. Whatever the reason, the reduction of these enzymes is not dependent on the growth conditions used, since both solid tumor and cultured tumor cells showed similar levels of activity. This is also seen in the outer membrane enzymes rotenoneinsensitive NADH-cyt c reductase, which was reduced 60% in both solid tumor and cultured tumor cell mitochondria, and monoamine oxidase, which was assayed with the use of 2 substrates. For each enzyme, the activity was 50-60% lower for both solid tumor and cultured tumor cell mitochondria. Reduced activity of this enzyme was also reported in poorly differentiated, fast-growing Morris hepatomas (18). Biochemical characterization of mitochondria from hamster melanoma cells and their comparison with hamster-embryo cell mitochondria again revealed modification of the normal enzyme complements in the neoplastic melanoma cells. Cytochrome oxidase and malate dehydrogenase activities were similar in melanoma and embryo cell mitochondria, but adenylate kinase, NADH-cyt c reductase, and NADH oxidase were reduced twofold to threefold in the melanoma organelles. Also, the outer membrane flavoprotein, rotenone-insensitive NADH-cyt c reductase, was reduced twofold in melanoma mitochondria. Uniquely, mitochondria from these neoplastic cells showed no activity when assayed for monoamine oxidase with the use of either 14Ctryptamine or 14C-serotonin as substrates. These assays are sensitive to the level of 0.01 nmole product/ minute/rug protein,'and we conclude that melanoma mitochondria have undergone almost a complete loss of this enzyme. Monoamine oxidase exists in multiple molecular forms in various tissues in many species (21-23). Possibly melanoma mitochondria have lost the isozymic forms of this enzyme that oxidize the substrates we tested but have retained other forms. This possiblity should be tested, but hamster-embryo cell mitochondria have significant activity toward the substrates tested, and loss of these isozymes in melanoma mitochondria represents a significant alteration in these organelles. This deviation and other abnormalities in the mitochondria of melanoma cells closely resemble the changes found in Novikoff hepatoma mitochondria. The mitochondrion as the site of cellular energy production and its central role in the control of a vast array of metabolic pathways make it important that this organelle is studied more intensively in a large variety of neoplastic cells.

6 558 WHITE, ARYA, AND TEWARI REFERENCES (1) WHITE MT, TEWARI KK: Structural and functional changes in Novikoff hepatoma mitochondria. Cancer Res 33: ,1973 (2) MOORE GE, LEHNER DR, KIKUCHI Y, et al: Continuous culture of melanotic cell line from the golden hamster. Science 137 :986, 1962 (3) FORTNER JG, MAHY AG, SCHRODT GA: Transplantable tumors of the Syrian (golden) hamsters. Cancer Res 21 : ,1961 (4) McALLISTER RM, RIGGsJL, REED G, et al: Transformation of rodent cells by simian adenovirus SA-7. Proc Soc Exp Bioi Med 131 : , 1969 (5) SMITH L: Spectrophotometric assay of cytochrome c oxidase. Methods Biochem Anal 2 : , 1955 (6) OCHOA S: Malic dehydrogenase from pig heart. Methods Enzymol I : , 1955 (7) SCHNAITMAN C, GREENAWALT JW: Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J Cell BioI 38: , 1968 (8) SOTTOCASA GL, KUYLENSTIERNA B, ERNSTER L, et al: An electron-transport system associated with the outer membrane of liver mitochondria. J Cell BioI 32: , 1967 (9) GREEN DE, ZIEGLER DM: Electron transport particles. Methods Enzymol 6: , 1963 (10) BONNER WP: Succinic dehydrogenase. Methods Enzymol 1: , 1955 (11) WURTMAN RJ, AXELROD JA: A sensitive and specific assay for the estimation of monoamine oxidase. Biochem PharmacoI12: ,1963 (12) MCCAMAN RE, MCCAMAN MW, HUNT JM, et al: Microdetermination of monoamine oxidase and 5 hydroxytryptophane decarboxylase activities in nervous tissues. J Neurochem 12: 15-23, 1965 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) LOWRY OH, ROSEBROUGH NJ, FARR AL, et al: Protein measurement with the protein phenol reagent. J Bioi Chern 193: ,1951 SPURR AR: A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26 :31-43, 1969 REYNOLDS ES: The use of lead citrate at high ph as an electron-opaque stain for electron microscopy. J Cell Bioi 17: , 1963 SCHNAITMAN C, ERWIN VG, GREENAWALT JW: The submitochondrial localization of monoamine oxidase. J Cell Bioi 32: ,1967 SCHREIBER JR, BALCAVAGE WX, MORRIS HP, et al: Enzymatic and spectral analysis of cytochrome oxidase in adult and fetal rat liver and Morris hepatoma 3924A. Cancer Res 30 : , 1970 PEDERSEN JB, GREENAWALT JW, CHAN TL, et al : Comparison of some ultrastructural and biochemical properties of mitochondria from Morris hepatomas 9618A, 7800A, and 3924A. Cancer Res 30: , 1970 CRISS WE: A review of isozyrnes in cancer. Cancer Res 31 : ,1971 CRISS WE, LITWACK G, MORRIS HP, et al: Adenosine triphosphate: Adenosine monophosphate phosphotransferase isozymes in rat liver and hepatomas. Cancer Res 30 : , 1970 YOUDIM MB: Multiple forms of mitochondrial monoamine oxidase. Br Med Bull 29: ,1973 TIPTON KF: Biochemical aspects of monoamine oxidase. Br Med Bull 29: ,1973 RAGLAND JB: Multiplicity of mitochondrial monoamine oxidases. Biochem Biophys Res Commun 31 : , 1968

7 FIGURE I.-Electron micrographs of thin sections of liver tissue, solid Novikoff hepatoma, and cultured hepatoma cells. a) normal liver, X 26,000; b) solid tumor, X 26,000; and c) cultured hepatoma cell, X 50,000. Preparations were fixed with glutaraldehyde and OS04 and stained with uranyl acetate and lead citrate. WHITE, ARYA, AND TEWARI 559