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[20] Neoplastic tissue mitochondria
110
PREPARATION OF MITOCHONDRIA
[20]
Comments
Grinding of ascites tumor cells with glass beads in the mortar and pestle (Method A) can be applied to small amounts of ceils, e.g., 1 ml of packed cells (140 mg of cell protein), if necessary. When more than 5 ml of packed cells is used, either a larger mortar can be used or the grinding procedure can be operated with 5 ml of packed cells in each run. Homogenization of tumor cells in a Dounce homogenizer (method B) is more applicable to experiments where larger amounts of mitochondria are needed. Mitochondria prepared by these two methods give high P: 0 ratio and respiratory control ratio, comparable to mitoehondria prepared from other tissue sources.
[ 20 ] Neoplastic Tissue Mitochondria
By THOMAS M. DEVLI~¢ The multiplicity of solid tumor types, originating from a variety of normal tissues, makes it impossible to generalize about the isolation, properties, and activities of mitoehondria from neoplasms. Even with one tumor type, variations in the amount of connective tissue, degree of necrosis, contamination by normal tissue, and lability of the mitochondria requires that each tumor line be considered as a different entity, with preparative and assay procedures tailored to the individual malignancy. H o m o g e n i z a t i o n and Isolation M e d i u m
With some tumors, isotonic sucrose (0.25 M) suffices for the isolation of active mitoehondria comparable to those from normal tissues. 1-3 This is particularly the case for soft tumors, such as hepatomas, where disruption of the cells occurs under mild homogenization2 Higher sucrose concentrations (e.g., 0.35 M) have been employed to yield mitochondria with latent ATPase from Murphy-Sturm rat lymphosarcoma. 4 The use of mannitol, polyvinylpyrrolidone, or raffinose has not been extensively tested, but may yield better preparations from some tumors. ~ Emmelot 1R. K. Kielley, Cancer Res. 12, 124 (1952). 2A. C. Aisenberg, Cancer Res. ~.1, 295 (1961); Cancer Res. 21, 304 (1961). 3G. E. Boxer and T. M. Devlin, Science 134, 1495 (1961). See also T. M. Devlin and M. P. Pruss, Proc. Am. Assoc. Cancer Res. 3, 315 (1962). M. Blecher and A. White, J. Biol. Chem. 235, 3404 (1960). 5p. Emmelot, C. J. Bos, P. J. Brombacher, and J. F. :Hampe, Brit. J. Cancer 13, 348 (1959).
[20]
M I T O C H O N D R IFROM A NEOPLASTIC TISSUE
111
have reported that inclusion of 0.001 M EDTA, pH 7.4, in 0.25 M sucrose yields preparations from some but not all tumors with low ATPase and high phosphorylating activity. It is generally advisable to include EDTA if the tissue is difficult to homogenize (Walker-256 carcinosarcoma, fibrosarcomas, and mammary carcinomas) or the mitochondria are extremely sensitive to aging (rapidly growing hepatomas). The inclusion of nicotinamide (0.02M) is beneficial in protecting the mitochondrial NAD from destruction by NADase 5,6 during isolation and assay. Isolated mitochondria from some tumors contain high NADase activity ~ presumably due to contamination by microsomes. 8 The low phosphorylating capacities of preparations from some neoplasms (e.g., Novikoff and Dunning hepatomas) can be stimulated by serum albumin in the incubation system2 In the author's laboratory, similar P:O ratios were obtained if the albumin (10 mg/ml) was present in the isolation medium or added to the assay system. e t al. 5
Isolation of Mitochondria The choice of experimental tumor depends on the objectives of the experiment and the availability of the tumor. Some mouse and rat tumors are in general use (e.g., Morris hepatomas, Novikoff hepatoma, Walker256, Sarcoma-180, adenocarcinoma-755, Murphy-Sturm lymphosarcoma, etc.) and are obtained easily. Many tumors are, however, only available in the laboratory where the tumor originated. The availability of the series of transplanted Morris hepatomas, 1° which vary in their degree of dedifferentiation and rate of growth, permit comparative studies of the activities of mitochondria from normal and neoplastic liver. The methods of isolation employed are variations of that described by Schneider and Hogeboom. 11 All steps are performed at 0-4 °. After excising the tumor, adjoining normal tissue and gross necrotic areas are removed, and the tumor is rinsed in cold homogenization medium to remove liquefied necrotic contamination and blood. In comparing yields of mitochondria from different tumors, histological examination is necessary to monitor the degree of contamination by normal tissue and to detect any major change in cell type. With tumors transplanted many L. A. Sauer, A. P. Martin, and E. Stotz, Cancer Res. 22, 632 (1962). 7p. Emmelot and P. J. Brombacher, Biochim. Biophys. Acta 22, 487 (1956). sp. Emmelot, Exptl. Cell Res. 13, 601 (1957). ' T. M. Devlin and M. P. Pruss, Federation Proc. 17, 211 (1958). Also T. M. Devlin, unpublished observations. '°H. P. Morris, Advan. Ca~wer Res. 9, 232 (1965). 'IW. C. Schneider and G. H. Hogeboom, J. Biol. Chem. 183, 123 (1950). See also G. H. Hogeboom, Vol. I [3].
112
PREPARATION OF MITOCHONDRIA
[20]
times and growing intraperitoneally this is not a stringent requirement. The tumor is blotted dry with filter paper and weighed. The tissue is disrupted with a Potter-Elvehjem type homogenizer, employing a Teflon pestle rotated at 2000 rpm; attempts to use other types of mechanical tissue homogenizers have been unsuccessful. After dicing the tissue, homogenization is performed using a ratio of 1 g tumor to 6-9 ml of medium. The minimum number of passes (10-20) is preferable. With soft tumors this should give nearly 100% cell disruption, but with tissues containing large amounts of connective tissue and stroma, the homogenization should be interrupted for recooling or preferably the undisrupted tissue should be rehomogenized in fresh medium. The homogenate, diluted to 10% (w/v), is filtered through two layers of cheesecloth to remove pieces of intact tissue and fibrous material. Nuclei and cell debris are sedimented by centrifugation at 600-700 g for 10 minutes. Extreme care should be employed in decanting the supernatant to exclude cell debris and intact erythrocytes. For maximum yields of mitochondria, the pellet can be resuspended (and rehomogenized, if necessary) and recentrifuged at 600 g; the supernatants are combined. In this laboratory, this step is omitted because it lengthens the time for isolation and the increase in the yield of mitochondria is usually small. Mitochondria can be sedimented at forces of 8000-12,000 g for 10 minutes; the higher centrifugal forces are often necessary because mitochondria from some tumors are smaller than those of normal tissues, but the possibility of contamination and damage to the mitochondria is also increased. Extreme care is employed in decantation or aspiration to completely remove the buffy coat. A lipid layer frequently observed at the top of the tube, should be completely removed by aspiration and wiping the inner wall of the tube with gauze. This lipid, if it contaminates subsequent steps, can lead to mitochondrial preparations with uncoupled oxidative phosphorylat~on, due to the presence of free fatty acids. 1= The mitochondrial pellet is resuspended with the use of a cold finger (thin test tube containing ice) by first making a smooth paste followed by the slow addition of medium. With this technique, clumps of agglutinated mit~chondria are rarely observed. The mitochondria are washed at least two times, employing the same centrifugal speeds as before and with the same volume as the original homogenate. This author has found that mitochondria washed only once are badly contaminated with soluble enzymes and microsomes, and their use leads to erroneous interpretations of the activities assayed. In many instances, three times washed mitochondria have been employed with *~P. Emmelot, Cancer Res. 22, 38 (1962).
[20]
MITOCHONDRIA FROM NEOPLASTIC TISSUE
113
excellent results. The total time of the isolation procedure should be kept to a minimum, each step being executed without delay; the mitochondria should be used immediately. Because of the varying yields from tumors, the final suspension of mitochondria must be adjusted to fit the needs of the experiment; suspensions containing the equivalent of 2.03.0 g of the original wet weight of tissue per milliliter usually yields a suspension containing 1-3 mg protein nitrogen per milliliter. Assay Systems Employing Neoplastic Tissue Mitochondria
The test systems for measurement of mitochondrial activities (e.g., respiration, phosphorylation, ATPase, swelling, and contraction) are essentially those employed for normal tissue mitochondria. :3 In contrast to liver mitochondria, exogenous cytochrome c (3 X 10-~M) and NAD (1.0 mM) are often required for maximum rates of respiration, particularly where long-term incubations (manometric studies) are performed. 1':'14,1~ Because of the higher ATPase activity of some neoplastic mitochondria, added fluoride (0.01 M) is often an absolute requirement for measurement of phosphorylation. Serum albumin (5-10 mg/ml) in the incubation medium also is employed to obtain maximum P : 0 ratios and latent ATPase activity with some tumors2 ,9 Nicotinamide (0.01-0.05 M) is beneficial if the preparations are contaminated with NADase. In shortterm experiments (polarographie measurement of respiration) these various cofactors are often unnecessary, since their addition is usually required to offset the effect of aging that occurs during long incubations. Properties of Neoplastic Mitochondria The properties and activities of mitochondria from malignant in comparison to normal tissues have been reviewed by Aisenberg, 16 and the reader is referred to this text for a complete description. It is now generally considered that most of the differences are quantitative rather than qualitative and that some of the alterations, such as higher ATPase and lower pyridine nucleotide content, may be due to changes occurring during isolation. ~6 The one general characteristic of neoplastic compared to normal tissue mitochondria is apparently their greater lability during isolation and storage. 1 Isolated mitochondria from a variety of neoplastic tissues manifest 13See this volume [3, 9, and 105b]. ~ H. G. Williams-Ashman and E. P. Kennedy, Cancer Res. 12, 415 (1952). 15C. E. Wenner and S. Weinhouse, Cancer Res. 13, 21 (1952). 16A. C. Aisenberg, "The Glycolysis and Respiration of Tumors." Academic Press, New York, 1961.
114
PREPARATION OF MITOCHONDRIA
[21]
respiratory rates and yield P : O ratios nearly equivalent to rat liver mitochondria. ~,~,~ The relative intactness of mitochondria from a series of rat hepatomas has been demonstrated, in the absence of added cofactors, as measured by their inability to oxidize exogenous NADH, low ATPase and high values of respiratory control2 Some degree of respiratory control for neoplastic tissue mitochondria has also been reported by Aisenberg 2 when measured manometrically, and values approaching three to five have been observed 3 when assayed polarographically. The difficulties in obtaining high P : O ratios in earlier studies ~4 as well as the inability of some tumors to oxidize fatty acids 5 can in most cases be attributed to their high ATPase activity. Phosphorylation in all cases is abolished by 2,4-dinitrophenol. It is of interest, that with some tumors manifesting phosphorylation, such as the Morris hepatoma 3683 and 3924, 2,4-dinitrophenol does not stimulate a significant ATPase2 This observation suggests the potential usefulness of mitochondria from neoplastic tissues in elucidating mechanisms sometimes obscured in normal tissues. Mitochondria from some malignant tissues, however, have been observed to yield very low P : 0 ratios, whereas other activities have been normal, suggesting some impairment in phosphorylating capacity2 Whether this is due to some subtle damage to the mitochondria during isolation or is actually a manifestation of the state of oxidative phosphorylation in the cell has yet to be determined.
[ 21 ] A T e c h n i q u e f o r t h e I s o l a t i o n of M i t o c h o n d r i a from Bovine Lymphocytes
By JOHN TERRANCEDAVIS In connection with a study in progress at this laboratory, it was necessary to develop a technique for isolating intact mitochondria from bovine lymphocytes. As it is almost impossible to obtain a relatively pure suspension of lymphocytes from peripheral blood, the lymph node was utilized as a source of lymphocytes for mitochondrial study. The procedure is essentially a modification of standard cellular fractionation procedures, 1 and consists of two steps: (1) the preparation of a suspension of lymphocytes from the nodes, and (2) the isolation of mitochondria from the suspension by standard techniques. 1See G. H. Hogeboom, Vol. I, p. 16.
PREPARATION OF MITOCHONDRIA
[20]
Comments
Grinding of ascites tumor cells with glass beads in the mortar and pestle (Method A) can be applied to small amounts of ceils, e.g., 1 ml of packed cells (140 mg of cell protein), if necessary. When more than 5 ml of packed cells is used, either a larger mortar can be used or the grinding procedure can be operated with 5 ml of packed cells in each run. Homogenization of tumor cells in a Dounce homogenizer (method B) is more applicable to experiments where larger amounts of mitochondria are needed. Mitochondria prepared by these two methods give high P: 0 ratio and respiratory control ratio, comparable to mitoehondria prepared from other tissue sources.
[ 20 ] Neoplastic Tissue Mitochondria
By THOMAS M. DEVLI~¢ The multiplicity of solid tumor types, originating from a variety of normal tissues, makes it impossible to generalize about the isolation, properties, and activities of mitoehondria from neoplasms. Even with one tumor type, variations in the amount of connective tissue, degree of necrosis, contamination by normal tissue, and lability of the mitochondria requires that each tumor line be considered as a different entity, with preparative and assay procedures tailored to the individual malignancy. H o m o g e n i z a t i o n and Isolation M e d i u m
With some tumors, isotonic sucrose (0.25 M) suffices for the isolation of active mitoehondria comparable to those from normal tissues. 1-3 This is particularly the case for soft tumors, such as hepatomas, where disruption of the cells occurs under mild homogenization2 Higher sucrose concentrations (e.g., 0.35 M) have been employed to yield mitochondria with latent ATPase from Murphy-Sturm rat lymphosarcoma. 4 The use of mannitol, polyvinylpyrrolidone, or raffinose has not been extensively tested, but may yield better preparations from some tumors. ~ Emmelot 1R. K. Kielley, Cancer Res. 12, 124 (1952). 2A. C. Aisenberg, Cancer Res. ~.1, 295 (1961); Cancer Res. 21, 304 (1961). 3G. E. Boxer and T. M. Devlin, Science 134, 1495 (1961). See also T. M. Devlin and M. P. Pruss, Proc. Am. Assoc. Cancer Res. 3, 315 (1962). M. Blecher and A. White, J. Biol. Chem. 235, 3404 (1960). 5p. Emmelot, C. J. Bos, P. J. Brombacher, and J. F. :Hampe, Brit. J. Cancer 13, 348 (1959).
[20]
M I T O C H O N D R IFROM A NEOPLASTIC TISSUE
111
have reported that inclusion of 0.001 M EDTA, pH 7.4, in 0.25 M sucrose yields preparations from some but not all tumors with low ATPase and high phosphorylating activity. It is generally advisable to include EDTA if the tissue is difficult to homogenize (Walker-256 carcinosarcoma, fibrosarcomas, and mammary carcinomas) or the mitochondria are extremely sensitive to aging (rapidly growing hepatomas). The inclusion of nicotinamide (0.02M) is beneficial in protecting the mitochondrial NAD from destruction by NADase 5,6 during isolation and assay. Isolated mitochondria from some tumors contain high NADase activity ~ presumably due to contamination by microsomes. 8 The low phosphorylating capacities of preparations from some neoplasms (e.g., Novikoff and Dunning hepatomas) can be stimulated by serum albumin in the incubation system2 In the author's laboratory, similar P:O ratios were obtained if the albumin (10 mg/ml) was present in the isolation medium or added to the assay system. e t al. 5
Isolation of Mitochondria The choice of experimental tumor depends on the objectives of the experiment and the availability of the tumor. Some mouse and rat tumors are in general use (e.g., Morris hepatomas, Novikoff hepatoma, Walker256, Sarcoma-180, adenocarcinoma-755, Murphy-Sturm lymphosarcoma, etc.) and are obtained easily. Many tumors are, however, only available in the laboratory where the tumor originated. The availability of the series of transplanted Morris hepatomas, 1° which vary in their degree of dedifferentiation and rate of growth, permit comparative studies of the activities of mitochondria from normal and neoplastic liver. The methods of isolation employed are variations of that described by Schneider and Hogeboom. 11 All steps are performed at 0-4 °. After excising the tumor, adjoining normal tissue and gross necrotic areas are removed, and the tumor is rinsed in cold homogenization medium to remove liquefied necrotic contamination and blood. In comparing yields of mitochondria from different tumors, histological examination is necessary to monitor the degree of contamination by normal tissue and to detect any major change in cell type. With tumors transplanted many L. A. Sauer, A. P. Martin, and E. Stotz, Cancer Res. 22, 632 (1962). 7p. Emmelot and P. J. Brombacher, Biochim. Biophys. Acta 22, 487 (1956). sp. Emmelot, Exptl. Cell Res. 13, 601 (1957). ' T. M. Devlin and M. P. Pruss, Federation Proc. 17, 211 (1958). Also T. M. Devlin, unpublished observations. '°H. P. Morris, Advan. Ca~wer Res. 9, 232 (1965). 'IW. C. Schneider and G. H. Hogeboom, J. Biol. Chem. 183, 123 (1950). See also G. H. Hogeboom, Vol. I [3].
112
PREPARATION OF MITOCHONDRIA
[20]
times and growing intraperitoneally this is not a stringent requirement. The tumor is blotted dry with filter paper and weighed. The tissue is disrupted with a Potter-Elvehjem type homogenizer, employing a Teflon pestle rotated at 2000 rpm; attempts to use other types of mechanical tissue homogenizers have been unsuccessful. After dicing the tissue, homogenization is performed using a ratio of 1 g tumor to 6-9 ml of medium. The minimum number of passes (10-20) is preferable. With soft tumors this should give nearly 100% cell disruption, but with tissues containing large amounts of connective tissue and stroma, the homogenization should be interrupted for recooling or preferably the undisrupted tissue should be rehomogenized in fresh medium. The homogenate, diluted to 10% (w/v), is filtered through two layers of cheesecloth to remove pieces of intact tissue and fibrous material. Nuclei and cell debris are sedimented by centrifugation at 600-700 g for 10 minutes. Extreme care should be employed in decanting the supernatant to exclude cell debris and intact erythrocytes. For maximum yields of mitochondria, the pellet can be resuspended (and rehomogenized, if necessary) and recentrifuged at 600 g; the supernatants are combined. In this laboratory, this step is omitted because it lengthens the time for isolation and the increase in the yield of mitochondria is usually small. Mitochondria can be sedimented at forces of 8000-12,000 g for 10 minutes; the higher centrifugal forces are often necessary because mitochondria from some tumors are smaller than those of normal tissues, but the possibility of contamination and damage to the mitochondria is also increased. Extreme care is employed in decantation or aspiration to completely remove the buffy coat. A lipid layer frequently observed at the top of the tube, should be completely removed by aspiration and wiping the inner wall of the tube with gauze. This lipid, if it contaminates subsequent steps, can lead to mitochondrial preparations with uncoupled oxidative phosphorylat~on, due to the presence of free fatty acids. 1= The mitochondrial pellet is resuspended with the use of a cold finger (thin test tube containing ice) by first making a smooth paste followed by the slow addition of medium. With this technique, clumps of agglutinated mit~chondria are rarely observed. The mitochondria are washed at least two times, employing the same centrifugal speeds as before and with the same volume as the original homogenate. This author has found that mitochondria washed only once are badly contaminated with soluble enzymes and microsomes, and their use leads to erroneous interpretations of the activities assayed. In many instances, three times washed mitochondria have been employed with *~P. Emmelot, Cancer Res. 22, 38 (1962).
[20]
MITOCHONDRIA FROM NEOPLASTIC TISSUE
113
excellent results. The total time of the isolation procedure should be kept to a minimum, each step being executed without delay; the mitochondria should be used immediately. Because of the varying yields from tumors, the final suspension of mitochondria must be adjusted to fit the needs of the experiment; suspensions containing the equivalent of 2.03.0 g of the original wet weight of tissue per milliliter usually yields a suspension containing 1-3 mg protein nitrogen per milliliter. Assay Systems Employing Neoplastic Tissue Mitochondria
The test systems for measurement of mitochondrial activities (e.g., respiration, phosphorylation, ATPase, swelling, and contraction) are essentially those employed for normal tissue mitochondria. :3 In contrast to liver mitochondria, exogenous cytochrome c (3 X 10-~M) and NAD (1.0 mM) are often required for maximum rates of respiration, particularly where long-term incubations (manometric studies) are performed. 1':'14,1~ Because of the higher ATPase activity of some neoplastic mitochondria, added fluoride (0.01 M) is often an absolute requirement for measurement of phosphorylation. Serum albumin (5-10 mg/ml) in the incubation medium also is employed to obtain maximum P : 0 ratios and latent ATPase activity with some tumors2 ,9 Nicotinamide (0.01-0.05 M) is beneficial if the preparations are contaminated with NADase. In shortterm experiments (polarographie measurement of respiration) these various cofactors are often unnecessary, since their addition is usually required to offset the effect of aging that occurs during long incubations. Properties of Neoplastic Mitochondria The properties and activities of mitochondria from malignant in comparison to normal tissues have been reviewed by Aisenberg, 16 and the reader is referred to this text for a complete description. It is now generally considered that most of the differences are quantitative rather than qualitative and that some of the alterations, such as higher ATPase and lower pyridine nucleotide content, may be due to changes occurring during isolation. ~6 The one general characteristic of neoplastic compared to normal tissue mitochondria is apparently their greater lability during isolation and storage. 1 Isolated mitochondria from a variety of neoplastic tissues manifest 13See this volume [3, 9, and 105b]. ~ H. G. Williams-Ashman and E. P. Kennedy, Cancer Res. 12, 415 (1952). 15C. E. Wenner and S. Weinhouse, Cancer Res. 13, 21 (1952). 16A. C. Aisenberg, "The Glycolysis and Respiration of Tumors." Academic Press, New York, 1961.
114
PREPARATION OF MITOCHONDRIA
[21]
respiratory rates and yield P : O ratios nearly equivalent to rat liver mitochondria. ~,~,~ The relative intactness of mitochondria from a series of rat hepatomas has been demonstrated, in the absence of added cofactors, as measured by their inability to oxidize exogenous NADH, low ATPase and high values of respiratory control2 Some degree of respiratory control for neoplastic tissue mitochondria has also been reported by Aisenberg 2 when measured manometrically, and values approaching three to five have been observed 3 when assayed polarographically. The difficulties in obtaining high P : O ratios in earlier studies ~4 as well as the inability of some tumors to oxidize fatty acids 5 can in most cases be attributed to their high ATPase activity. Phosphorylation in all cases is abolished by 2,4-dinitrophenol. It is of interest, that with some tumors manifesting phosphorylation, such as the Morris hepatoma 3683 and 3924, 2,4-dinitrophenol does not stimulate a significant ATPase2 This observation suggests the potential usefulness of mitochondria from neoplastic tissues in elucidating mechanisms sometimes obscured in normal tissues. Mitochondria from some malignant tissues, however, have been observed to yield very low P : 0 ratios, whereas other activities have been normal, suggesting some impairment in phosphorylating capacity2 Whether this is due to some subtle damage to the mitochondria during isolation or is actually a manifestation of the state of oxidative phosphorylation in the cell has yet to be determined.
[ 21 ] A T e c h n i q u e f o r t h e I s o l a t i o n of M i t o c h o n d r i a from Bovine Lymphocytes
By JOHN TERRANCEDAVIS In connection with a study in progress at this laboratory, it was necessary to develop a technique for isolating intact mitochondria from bovine lymphocytes. As it is almost impossible to obtain a relatively pure suspension of lymphocytes from peripheral blood, the lymph node was utilized as a source of lymphocytes for mitochondrial study. The procedure is essentially a modification of standard cellular fractionation procedures, 1 and consists of two steps: (1) the preparation of a suspension of lymphocytes from the nodes, and (2) the isolation of mitochondria from the suspension by standard techniques. 1See G. H. Hogeboom, Vol. I, p. 16.