- Email: [email protected]
[66] Use of mammary gland tissue for the study of polyamine metabolism and function
[66]
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tors in this experiment. As has been shown with methylglyoxal bis(guanylhydrazone) and spermidine, 2 there may be competition for entry into the cells between the drug and the added polyamine. A second complicating factor is that newborn calf serum, commonly used in culturing the cells, contains high levels of an amine oxidase active against spermidine and spermine21; the oxidized products of these polyamines are highly toxic to animal cells. 22-24 Therefore, prior to addition of exogenous polyamines, the cells must be placed either in medium containing horse serum, J9which is low in the amine oxidase 24,25 or in a serum-free medium. 2° 3. Delaying the addition of the drug, to allow limited polyamine accumulation prior to inhibition, should show a relationship between polyamine levels and the physiological effect. 4. The physiological response of the cells to structural variants of the drug in question should be related to their effectiveness in inhibiting polyamine biosynthesis. This has been tested in the lymphocyte system for analogs of methylglyoxal bis(guanylhydrazone).14 In the case of the ornithine analogs, relatively high concentrations are sometimes required to inhibit polyamine biosynthesis in vivo. An important control, that has been suggested, 26 is to test DE- or L-ornithine at the concentration of the ornithine analog used. In the case of 9L rat brain tumor cells, 50 mM ornithine produced the same effect as 50 mM a-methylornithine on cell proliferation, with no influence on intracellular polyamine content. 26 21 j. G. Hirsch, J. Exp. Med. 97, 345 (1953). 22 R. A. Alarcon, G. E. Foley, and E. J. Modest, Arch. Biochem. Biophys. 94, 540 (1961). 23 U. Bachrach, S. Abzug, and A. Bekierkunst, Biochim. Biophys. Acta 134, 174 (1967). 24 M. L. Higgins, M. C. Tillman, J. P. Rupp, and F. R. Leach, J. Cell. Physiol. 74, 149 (1969). 25 H. Blaschko, Adv. Comp. Physiol. Biochem. 1, 67 (1962). 26 j. Seidenfeld and L. J. Marton, in " P o l y a m i n e s in Biology and Medicine" (D. R. Morris and L. J. Marton, eds.), p. 311. Dekker, N e w York, 1981.
[66] U s e o f M a m m a r y G l a n d T i s s u e for t h e S t u d y o f Polyamine Metabolism and Function By TAKAMI OKA and JOHN W. PERRY The mammary gland provides a useful and unique model for the study of polyamine metabolism and function in the hormonal induction of cell growth and differentiation. The growth and development of the mammary gland are virtually arrested in the adult, nonpregnant state. During pregMETHODS IN ENZYMOLOGY,VOL. 94
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
390
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[66]
nancy mammary epithelial cells undergo extensive proliferation to form a network of lobuloalveolar cells that synthesize the characteristic milk components during the period of lactation. The concentrations of polyamines in mammary cells begin to rise during pregnancy when cell proliferation occurs and reach maximal levels during the lactation period, when milk proteins are formed. J.2 The activities of several enzymes involved in the biosynthesis of polyamines also increase markedly during these periods. 1,2 The morphological and biochemical changes that occur during pregnancy and lactation can be induced in culture by cultivating mouse mammary tissue explants in a chemically defined synthetic medium containing appropriate combinations of hormones. 3 When mammary tissue explants are cultured in the presence of insulin, the mammary epithelium undergoes proliferation. The addition of glycocorticoid with insulin promotes development of cellular organelles such as rough endoplasmic reticulum. Under the influence of insulin, cortisol, and prolactin, mammary epithelial cells express their differentiative function by production of the milk proteins casein and a-lactalbumin. Because this organ culture system provides a well-controlled environment and is free of certain problems that are inherent in experiments performed in vivo, it has proved to be useful for delineating the complex regulatory mechanism of the development of the mammary gland. This chapter describes the various experimental procedures employed in the study of polyamine biosynthesis and function in the hormone-induced development of the mammary gland in culture.
Organ Culture of the Mammary Gland The animals were killed by cervical dislocation, and the abdominal mammary glands were removed bilaterally. Tissue explants weighing approximately 1 mg each were prepared by cutting tissue at 25 ° in Medium 199 (Hanks' salts) and cultured as described previously. 4 All procedures were done under sterile conditions. Insulin (crystalline pork zinc insulin, Eli Lilly) and bovine (or ovine) prolactin (NIH) were added to the medium at a final concentration of 5/zg/ml. Cortisol was used at a concentration of 1 T. Oka, T. Sakai, D. W. Lundgren, and J. W. Perry, in "Hormones, Receptors and Breast Cancer" (W. L. McGuire, ed), p. 301. Raven, New York, 1978. 2 D. H. Russell and T. McVicker, Biochem. J. 130, 71 (1972). 3 y . j. Topper and T. Oka, in "Lactation" (G. L. Larson and V. R. Smith, eds.), p. 327. Academic Press, New York, 1974. 4 y . j. Topper, T. Oka, and B. K. Vonderhaar, this series, Vol. 39, p. 443.
[66]
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0.03/.~M in the study of a-lactalbumin production and 3/xM for the induction of casein synthesis. 5 Assay of the Activity of Enzymes Involved in Polyamine Biosynthesis The activity of ornithine decarboxylase was determined by measuring the liberation of 14CO2 from DL-[1-14C]ornithine. Tissue explants were blotted, weighed, and homogenized at 4° in a small glass homogenizer (Kontes, ! ml capacity) with 0.3-0.4 ml of a solution containing 50 mM Tris-HC1, pH 7.5, 4 mM EDTA, 5 mM dithiothreitol, and 40/zM pyridoxal phosphate. The homogenate was centrifuged at 105,000 g for 60 min at 4°, and the supernatant fraction was removed for the enzyme assay. This fraction, which could be kept frozen at -20 ° for 2-3 days without any detectable loss of activity, contained virtually all the enzyme activity present in the homogenate. The amount of tissue explants used for the assay varied from 10 to 15 mg in the case of tissue from midpregnant mice to about 50 mg of virgin mouse tissue because of the variation in the basal level of the enzyme activity at different physiological states of the animals. Because enzyme activity in the mammary tissue can be altered by other factors such as osmolarity of the culture medium, 6 temperature during the preparation of explants, and the circadian rhythm of the animals, care must be taken to use consistent procedures in culture experiments. The enzyme assay was carried out in 25-ml Erlenmeyer flasks that contained 2 ml of the following reaction mixture: 5 mM dithiothreitol, 40 /xM pyridoxal phosphate, 4 mM EDTA, 50 mM Tris-HC1 (pH 7.5), 1/zM DL-[ 1-14C]ornithine (specific activity 8 mCi/mmol), and 0.1 to 0.2 ml of the enzyme solution. The DL-[1-14C]ornithine obtained from commercial sources was dissolved in 1 ml of 0.01 N HC1 and evaporated to dryness in a rotary evaporator before use. This procedure eliminated the 14CO2, which is apparently a contaminant in the original commercial preparation, and reduced the blank value in the assay below 80 cpm. A blank contained an equal amount of the reaction mixture without the enzyme solution. After addition of the reaction mixture and the enzyme, the flasks were closed with a rubber cap from which a glass well was suspended and shaken in a water bath at 37° for 60 min. Incubations were terminated by injecting 0.2 ml of Hyamine solution into the center glass well and 0.5 ml of 5 N H2SO4 into the incubation medium. Details of the procedure for trapping and counting ~4CO2 are given elsewhere. 7 Enzyme activity is 5 M. Ono and T. Oka, Cell 19, 473 (1980). 6 j. W. Perry and T. Oka, Biochim. Biophys. Acta 629, 24 (1980). 7 T. Oka and J. W. Perry, J. Biol. Chem. 251, 1738 (1976).
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[66]
expressed as picomoles of 14C02formed per hour per milligram of tissue explants. The activity of S-adenosyl-L-methionine decarboxylase was assayed by measuring ~4COzrelease from carboxyl-labeled substrate. The preparation of enzyme extract from explants was essentially identical to that described for ornithine decarboxylase, except that about 100 mg of tissue explants were homogenized in 0.5 ml of a solution containing 25 mM sodium phosphate buffer, pH 7.6, 1 mM dithiothreitol, 0.l mM EDTA, and 0.5 mM putrescine. The enzyme reaction was carried out in a total volume of 2 ml consisting of 0.1-0.2 ml of enzyme solution and the homogenizing buffer, which contained, per milliliter, 0.05 t~Ci of adenosyl[carboxy-t4C]methionine (specific activity 55 mCi/mmol). Other details of the procedure were essentially the same as those described for ornithine decarboxylase. Enzyme activity was expressed as picomoles of ~4CO2 formed per hour per milligram of tissue explants. The enzyme activity in mammary epithelium of cultured tissue is stimulated by the synergistic activity of insulin and glucocorticoid. 8 The enzyme from lactating mouse mammary gland has been purified to apparent homogeneity .9 An antibody to the pure enzyme was used in measuring levels of the enzyme in the extract of mammary tissue. 9 The activity of spermidine synthase was assayed by measuring the production of spermidine. Enzyme extract from mammary explants was prepared by homogenizing 50-100 mg of explants at 4° in 0.5 ml of 25 mM phosphate buffer, pH 7.2, containing 1 mM dithiothreitol and 0.1 mM EDTA. The homogenate was centrifuged for 60 min at 105,000 g at 4°, and the resultant supernatant solution was used for the enzyme assay. The reaction was carried out at 37° for 60 min in test tubes that contained 25 mM sodium phosphate buffer (pH 7.6), 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM decarboxylated adenosylmethionine, 1 mM [1,4~4C]putrescine (specific activity, 55 mCi/mmol), and 0. l ml of the enzyme solution in a volume of 0.15 ml as described previously. ~° A blank contained an equal amount of the reaction mixture without enzyme. Decarboxylated adenosylmethionine was synthesized enzymatically by the method of Tabor ~ with E. coli S-adenosylmethionine decarboxylase purified through step 4 by the method of Wickner et al. ~ The activity was s T. Oka and J. W. Perry, J. Biol. Chem. 249, 7674 (1974). 9 T. Sakai, C. Hori, K. K a n o , and T. Oka, Biochemistry 18, 5541 (1979). 10 j. J~inne, A. Schenone, and H. G. Williams-Ashman, Biochem. Biophys. Res. Commun. 42, 758 (1971). 1i C. W. Tabor, this series, Vol. 5, p. 754. 12 R. B. Wickner, C. W. Tabor, and H. Tabor, J. Biol. Chem. 245, 2132 (1970).
[66]
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expressed as picomoles of spermidine formed per hour per milligram of tissue explant. 13 The increase in enzyme activity in cultured explants is dependent on the presence of insulin and glucocorticoid. 13,14 The activity of arginase in mammary explants was determined by measuring the formation of urea from arginine as described earlier. 15 About 20-30 mg of explants were homogenized in 1 ml of distilled water, and the homogenate was treated at 55° for 5 min in the presence of 0.01 M MnCI2. The assay was initiated by the addition of 1 ml of 0.25 M Larginine (pH 9.7) and 0.01 ml of 0.1 M MnC12. After incubation at 37° for 15 min, the reaction was stopped by the addition of 2 ml of 6% (w/v) trichloroacetic acid, and the protein precipitate was removed by centrifugation. The amount of urea formed was determined colorimetrically j5 using a standard curve constructed with known concentrations of urea. Activity was expressed as micrograms of urea formed per 10 min per milligram wet weight of tissue. 16 The synergistic action of insulin and prolactin enhances the enzyme activity in cultured tissue.16 In the mammary gland of virgin and pregnant mice, mammary epithelial cells are surrounded by a considerable number of fat cells. To study the effects of hormones on enzyme activity in the epithelium of mammary explants, it is important to distinguish the response of epithelial cells from that of fat cells. This can be achieved by determining enzyme activity both in whole explants and in the isolated epithelial cells derived from the mammary explants of the parallel culture. The differential response of epithelial and fat cells to hormones was observed in the study of several enzymes involved in polyamine biosynthesis in cultured mammary tissue.7,8,13 Determination of Polyamine Content The content of putrescine, spermidine, and spermine in mammary explants was quantitated with ninhydrin using an amino acid analyzer) 7 About 50-100 mg of explants were homogenized in 1 ml of 3% perchloric acid. After centrifugation, the residue was washed by another 1 ml of 3% perchloric acid, and the combined supernatant fractions were neutralized with 4 N KOH. The precipitate was removed by centrifugation, and the 13 T. 14 T. 15 R. 16T. 17 D. 81
Oka, J. W. Perry, and K. Kano, Biochem. Biophys. Res. Commun. 79, 979 (1977). Sakai, D. W. Lundgren, and T. Oka, J. Cell. Physiol. 95, 259 (1978). T. Schimke, J. Biol. Chem. 39, 3809 (1964). Oka and J. W. Perry, Nature (London) 250, 660 (1974). W. Lundgren, P. M. Farrell, and P. A. di Sant'Agnese, Proc. Soc. Exp. Biol. Med. 152, (1976).
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supernatant solution was lyophilized. The dried residue was dissolved in a potassium chloride-potassium citrate buffer (2.34 M K+), pH 5.64, and assayed for the amount of polyamine. ~4 This method is more sensitive than the high-voltage electrophoretic method used in earlier studies. 8 In cultured mammary tissue from virgin mice, insulin increases the content of putrescine and spermidine about threefold over the initial level during a 3-day culture, whereas the content of spermine remains relatively unchanged. ~4 In culture of mammary tissue from midpregnant mice, the synergistic action of insulin, cortisol, and prolactin increases the concentration of spermidine in mammary epithelium about threefold in 2 days. 8 The combination of insulin and prolactin or the combination of insulin and cortisol enhances the polyamine level to a small extent. 8 The concentration of spermine in mammary epithelium increased approximately 30% during 2 days, but no appreciable difference was found among various combinations of the three hormones. 8 Uptake of Polyamines Mouse mammary gland has been shown to possess a transport system for spermidine, spermine, and putrescine.lS In order to measure the uptake of polyamine, 10-20 mg of explants were incubated in Medium 199 with appropriately labeled polyamines. The amount of radioactive polyamine added to the culture was 1-2 /zCi/ml and 0.01 /zCi/ml for 3Hlabeled and for 14C-labeled polyamine, respectively. Tritium- or 14C-labeled polyamines (specific activity 100-1000 mCi/mmol and 5-20 mCi/mmol, respectively) were obtained from commercial sources, checked for purity, and, whenever necessary, purified by high-voltage electrophoresis prior to use. s After addition of labeled polyamine, explants were harvested at various times, weighed, and placed for washing on a Whatman GS/C disk filter paper that had been immersed for at least 15 rain in Medium 199 containing 40 mM corresponding unlabeled polyamine to prevent adsorption of the residual external radioactive polyamines. Explants were washed on a Millipore filter apparatus with 15 ml of Medium 199 and 4 ml of phosphate-buffered 0.15 M NaCI (pH 7.2), using suction. Each washing solution also contained the appropriate unlabeled polyamine at 40 mM. This washing procedure removed approximately 10% of the radioactivity associated with explants, which presumably represented nonspecific adsorption of the polyamines to the cells. The washed explants on the filters were transferred into a scintillation vial, digested with 1 ml of tissue solubilizer, and assayed for radioactivity is K. Kano and T. Oka, J. Biol. Chem. 251, 2795 (1976).
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in a toluene-based scintillation fluid with a liquid scintillation spectrometer. Insulin stimulates the uptake of polyamines by enhancing Vmax for polyamine influx and preventing efflux of polyamine. ~8 Prolactin, in the presence of insulin, elicits a greater increase in Vmaxfor polyamine uptake. 18 Assessment of the Function of Polyamines in the Mammary Gland Hormonal stimulation of the mammary gland in culture causes marked enhancement of polyamine accumulation prior to acceleration of DNA synthesis ~4and milk-protein synthesis. 8 Since the mammary epithelium in virgin mice is essentially nonproliferative, but can be induced to undergo DNA synthesis in culture with insulin, this system is suited for examining the role of polyamines in the proliferation of mammary epithelium. On the other hand, the mammary cells in pregnant mice are in an active phase of proliferation and can be readily stimulated to undergo differentiation. Thus organ culture of pregnant mouse mammary tissue provides a system to study the function of polyamines in mammary differentiation. The biological importance of polyamine accumulation in the development of the mammary gland can be assessed by employing various culture conditions in which the intracellular level of polyamines is varied by alterations in hormone combination, 8 osmolarity, 6 amino acid composition of culture medium, ~6use of several inhibitors of ornithine decarboxylase, 19 and S-adenosylmethionine decarboxylase, 8 and finally, by use of mammary explants derived from virgin and pregnant mice containing initially a low and a high level of polyamines.~4,~9 It is also possible to change the intracellular concentration of polyamines by exogenously adding them to culture. 8,~4 Combinations of these experimental approaches have yielded some useful information concerning the role of polyamines in the development of the mammary gland. In cultured mammary explants from virgin mice, both putrescine and spermidine appear to be important for hormonal induction of DNA synthesis in mammary epithelium. 14,~9Furthermore, several lines of evidence suggest that spermidine (0.04 raM) can simulate the action of glucocorticoid on induction of a-lactalbumin synthesis in cultured tissue from midpregnant mice. 8 It is noteworthy that stimulation of synthesis of milk protein in rabbit mammary tissue can occur in the presence of only insulin and prolactin and does not require the action of glucocorticoid and spermidine. 2° On the other hand, synthesis of milk protein in rat mammary 19 H. Inoue and T. Oka, J. Biol. Chem. 255, 3308 (1980). 20 L. M. Houdebine, E. Devinoy, and C. Delouis, Biochimie 60, 735 (1978).
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gland is dependent on both glucocorticoid and spermidine, but spermidine, in place of cortisol, is unable to act synergistically with insulin and prolactin. 21 The reason for the observed species differences remains unknown. It should be emphasized that some caution must be exercised in interpreting these experimental results obtained by use of enzyme inhibitors and supraphysiological conditions because of other possible side effects. 21 F. F. Bolander and Y. J. Topper, Biochem. Biophys. Res. Commun. 90, 1131 (1979).
[67] O r n i t h i n e D e c a r b o x y l a s e A s s a y P e r m i t t i n g E a r l y D e t e r m i n a t i o n o f H i s t o c o m p a t i b i l i t y in t h e M i x e d Lymphocyte Reaction By
ACHILLES
A.
D E M E T R I O U , CELIA W H I T E TABOR,
a n d HERBERT TABOR
The rejection of transplanted organs is a consequence of genetic disparity between donor and recipient. The human major histocompatibility complex (MHC), termed HLA, is localized on chromosome six and it includes three loci (A, B, C) whose products can be recognized by complement-dependent lymphocytotoxicity techniques. Another important component is the HLA-D locus, whose products are recognizable by the mixed lymphocyte culture (MLC) test. In the MLC test, lymphocytes from a donor and a recipient are mixed in vitro, and antigenic determinants coded by the HLA-D region, which are present on the cell surface, provoke a mixed lymphocyte reaction of proliferation and blast transformation when genetically dissimilar lymphocytes interact. At present, the most widely used assay of mixed lymphocyte reactivity measures the incorporation of tritiated thymidine into DNA and requires 4-5 days. 1 This long waiting period has hampered the application of this technique to the prospective selection of recipients for cadaver organ transplantation. Changes in the activity of ornithine decarboxylase, at 18 hr after mixing in the MLC, can be used as an early, sensitive indicator of the degree of histocompatibility.2 1 F. H. Bach and K. Hirschhorn, Science 142, 813 (1964). 2 A. A. Demetriou, M. W. Flye, C. W. Tabor, and H. Tabor, Transplantation 27, 190 (1979).
METHODS IN ENZYMOLOGY,VOL. 94
Copyright © 1983by Academic Press, inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
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tors in this experiment. As has been shown with methylglyoxal bis(guanylhydrazone) and spermidine, 2 there may be competition for entry into the cells between the drug and the added polyamine. A second complicating factor is that newborn calf serum, commonly used in culturing the cells, contains high levels of an amine oxidase active against spermidine and spermine21; the oxidized products of these polyamines are highly toxic to animal cells. 22-24 Therefore, prior to addition of exogenous polyamines, the cells must be placed either in medium containing horse serum, J9which is low in the amine oxidase 24,25 or in a serum-free medium. 2° 3. Delaying the addition of the drug, to allow limited polyamine accumulation prior to inhibition, should show a relationship between polyamine levels and the physiological effect. 4. The physiological response of the cells to structural variants of the drug in question should be related to their effectiveness in inhibiting polyamine biosynthesis. This has been tested in the lymphocyte system for analogs of methylglyoxal bis(guanylhydrazone).14 In the case of the ornithine analogs, relatively high concentrations are sometimes required to inhibit polyamine biosynthesis in vivo. An important control, that has been suggested, 26 is to test DE- or L-ornithine at the concentration of the ornithine analog used. In the case of 9L rat brain tumor cells, 50 mM ornithine produced the same effect as 50 mM a-methylornithine on cell proliferation, with no influence on intracellular polyamine content. 26 21 j. G. Hirsch, J. Exp. Med. 97, 345 (1953). 22 R. A. Alarcon, G. E. Foley, and E. J. Modest, Arch. Biochem. Biophys. 94, 540 (1961). 23 U. Bachrach, S. Abzug, and A. Bekierkunst, Biochim. Biophys. Acta 134, 174 (1967). 24 M. L. Higgins, M. C. Tillman, J. P. Rupp, and F. R. Leach, J. Cell. Physiol. 74, 149 (1969). 25 H. Blaschko, Adv. Comp. Physiol. Biochem. 1, 67 (1962). 26 j. Seidenfeld and L. J. Marton, in " P o l y a m i n e s in Biology and Medicine" (D. R. Morris and L. J. Marton, eds.), p. 311. Dekker, N e w York, 1981.
[66] U s e o f M a m m a r y G l a n d T i s s u e for t h e S t u d y o f Polyamine Metabolism and Function By TAKAMI OKA and JOHN W. PERRY The mammary gland provides a useful and unique model for the study of polyamine metabolism and function in the hormonal induction of cell growth and differentiation. The growth and development of the mammary gland are virtually arrested in the adult, nonpregnant state. During pregMETHODS IN ENZYMOLOGY,VOL. 94
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
390
POLYAMINES IN LYMPHOCYTES AND MAMMARY GLAND
[66]
nancy mammary epithelial cells undergo extensive proliferation to form a network of lobuloalveolar cells that synthesize the characteristic milk components during the period of lactation. The concentrations of polyamines in mammary cells begin to rise during pregnancy when cell proliferation occurs and reach maximal levels during the lactation period, when milk proteins are formed. J.2 The activities of several enzymes involved in the biosynthesis of polyamines also increase markedly during these periods. 1,2 The morphological and biochemical changes that occur during pregnancy and lactation can be induced in culture by cultivating mouse mammary tissue explants in a chemically defined synthetic medium containing appropriate combinations of hormones. 3 When mammary tissue explants are cultured in the presence of insulin, the mammary epithelium undergoes proliferation. The addition of glycocorticoid with insulin promotes development of cellular organelles such as rough endoplasmic reticulum. Under the influence of insulin, cortisol, and prolactin, mammary epithelial cells express their differentiative function by production of the milk proteins casein and a-lactalbumin. Because this organ culture system provides a well-controlled environment and is free of certain problems that are inherent in experiments performed in vivo, it has proved to be useful for delineating the complex regulatory mechanism of the development of the mammary gland. This chapter describes the various experimental procedures employed in the study of polyamine biosynthesis and function in the hormone-induced development of the mammary gland in culture.
Organ Culture of the Mammary Gland The animals were killed by cervical dislocation, and the abdominal mammary glands were removed bilaterally. Tissue explants weighing approximately 1 mg each were prepared by cutting tissue at 25 ° in Medium 199 (Hanks' salts) and cultured as described previously. 4 All procedures were done under sterile conditions. Insulin (crystalline pork zinc insulin, Eli Lilly) and bovine (or ovine) prolactin (NIH) were added to the medium at a final concentration of 5/zg/ml. Cortisol was used at a concentration of 1 T. Oka, T. Sakai, D. W. Lundgren, and J. W. Perry, in "Hormones, Receptors and Breast Cancer" (W. L. McGuire, ed), p. 301. Raven, New York, 1978. 2 D. H. Russell and T. McVicker, Biochem. J. 130, 71 (1972). 3 y . j. Topper and T. Oka, in "Lactation" (G. L. Larson and V. R. Smith, eds.), p. 327. Academic Press, New York, 1974. 4 y . j. Topper, T. Oka, and B. K. Vonderhaar, this series, Vol. 39, p. 443.
[66]
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0.03/.~M in the study of a-lactalbumin production and 3/xM for the induction of casein synthesis. 5 Assay of the Activity of Enzymes Involved in Polyamine Biosynthesis The activity of ornithine decarboxylase was determined by measuring the liberation of 14CO2 from DL-[1-14C]ornithine. Tissue explants were blotted, weighed, and homogenized at 4° in a small glass homogenizer (Kontes, ! ml capacity) with 0.3-0.4 ml of a solution containing 50 mM Tris-HC1, pH 7.5, 4 mM EDTA, 5 mM dithiothreitol, and 40/zM pyridoxal phosphate. The homogenate was centrifuged at 105,000 g for 60 min at 4°, and the supernatant fraction was removed for the enzyme assay. This fraction, which could be kept frozen at -20 ° for 2-3 days without any detectable loss of activity, contained virtually all the enzyme activity present in the homogenate. The amount of tissue explants used for the assay varied from 10 to 15 mg in the case of tissue from midpregnant mice to about 50 mg of virgin mouse tissue because of the variation in the basal level of the enzyme activity at different physiological states of the animals. Because enzyme activity in the mammary tissue can be altered by other factors such as osmolarity of the culture medium, 6 temperature during the preparation of explants, and the circadian rhythm of the animals, care must be taken to use consistent procedures in culture experiments. The enzyme assay was carried out in 25-ml Erlenmeyer flasks that contained 2 ml of the following reaction mixture: 5 mM dithiothreitol, 40 /xM pyridoxal phosphate, 4 mM EDTA, 50 mM Tris-HC1 (pH 7.5), 1/zM DL-[ 1-14C]ornithine (specific activity 8 mCi/mmol), and 0.1 to 0.2 ml of the enzyme solution. The DL-[1-14C]ornithine obtained from commercial sources was dissolved in 1 ml of 0.01 N HC1 and evaporated to dryness in a rotary evaporator before use. This procedure eliminated the 14CO2, which is apparently a contaminant in the original commercial preparation, and reduced the blank value in the assay below 80 cpm. A blank contained an equal amount of the reaction mixture without the enzyme solution. After addition of the reaction mixture and the enzyme, the flasks were closed with a rubber cap from which a glass well was suspended and shaken in a water bath at 37° for 60 min. Incubations were terminated by injecting 0.2 ml of Hyamine solution into the center glass well and 0.5 ml of 5 N H2SO4 into the incubation medium. Details of the procedure for trapping and counting ~4CO2 are given elsewhere. 7 Enzyme activity is 5 M. Ono and T. Oka, Cell 19, 473 (1980). 6 j. W. Perry and T. Oka, Biochim. Biophys. Acta 629, 24 (1980). 7 T. Oka and J. W. Perry, J. Biol. Chem. 251, 1738 (1976).
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[66]
expressed as picomoles of 14C02formed per hour per milligram of tissue explants. The activity of S-adenosyl-L-methionine decarboxylase was assayed by measuring ~4COzrelease from carboxyl-labeled substrate. The preparation of enzyme extract from explants was essentially identical to that described for ornithine decarboxylase, except that about 100 mg of tissue explants were homogenized in 0.5 ml of a solution containing 25 mM sodium phosphate buffer, pH 7.6, 1 mM dithiothreitol, 0.l mM EDTA, and 0.5 mM putrescine. The enzyme reaction was carried out in a total volume of 2 ml consisting of 0.1-0.2 ml of enzyme solution and the homogenizing buffer, which contained, per milliliter, 0.05 t~Ci of adenosyl[carboxy-t4C]methionine (specific activity 55 mCi/mmol). Other details of the procedure were essentially the same as those described for ornithine decarboxylase. Enzyme activity was expressed as picomoles of ~4CO2 formed per hour per milligram of tissue explants. The enzyme activity in mammary epithelium of cultured tissue is stimulated by the synergistic activity of insulin and glucocorticoid. 8 The enzyme from lactating mouse mammary gland has been purified to apparent homogeneity .9 An antibody to the pure enzyme was used in measuring levels of the enzyme in the extract of mammary tissue. 9 The activity of spermidine synthase was assayed by measuring the production of spermidine. Enzyme extract from mammary explants was prepared by homogenizing 50-100 mg of explants at 4° in 0.5 ml of 25 mM phosphate buffer, pH 7.2, containing 1 mM dithiothreitol and 0.1 mM EDTA. The homogenate was centrifuged for 60 min at 105,000 g at 4°, and the resultant supernatant solution was used for the enzyme assay. The reaction was carried out at 37° for 60 min in test tubes that contained 25 mM sodium phosphate buffer (pH 7.6), 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM decarboxylated adenosylmethionine, 1 mM [1,4~4C]putrescine (specific activity, 55 mCi/mmol), and 0. l ml of the enzyme solution in a volume of 0.15 ml as described previously. ~° A blank contained an equal amount of the reaction mixture without enzyme. Decarboxylated adenosylmethionine was synthesized enzymatically by the method of Tabor ~ with E. coli S-adenosylmethionine decarboxylase purified through step 4 by the method of Wickner et al. ~ The activity was s T. Oka and J. W. Perry, J. Biol. Chem. 249, 7674 (1974). 9 T. Sakai, C. Hori, K. K a n o , and T. Oka, Biochemistry 18, 5541 (1979). 10 j. J~inne, A. Schenone, and H. G. Williams-Ashman, Biochem. Biophys. Res. Commun. 42, 758 (1971). 1i C. W. Tabor, this series, Vol. 5, p. 754. 12 R. B. Wickner, C. W. Tabor, and H. Tabor, J. Biol. Chem. 245, 2132 (1970).
[66]
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expressed as picomoles of spermidine formed per hour per milligram of tissue explant. 13 The increase in enzyme activity in cultured explants is dependent on the presence of insulin and glucocorticoid. 13,14 The activity of arginase in mammary explants was determined by measuring the formation of urea from arginine as described earlier. 15 About 20-30 mg of explants were homogenized in 1 ml of distilled water, and the homogenate was treated at 55° for 5 min in the presence of 0.01 M MnCI2. The assay was initiated by the addition of 1 ml of 0.25 M Larginine (pH 9.7) and 0.01 ml of 0.1 M MnC12. After incubation at 37° for 15 min, the reaction was stopped by the addition of 2 ml of 6% (w/v) trichloroacetic acid, and the protein precipitate was removed by centrifugation. The amount of urea formed was determined colorimetrically j5 using a standard curve constructed with known concentrations of urea. Activity was expressed as micrograms of urea formed per 10 min per milligram wet weight of tissue. 16 The synergistic action of insulin and prolactin enhances the enzyme activity in cultured tissue.16 In the mammary gland of virgin and pregnant mice, mammary epithelial cells are surrounded by a considerable number of fat cells. To study the effects of hormones on enzyme activity in the epithelium of mammary explants, it is important to distinguish the response of epithelial cells from that of fat cells. This can be achieved by determining enzyme activity both in whole explants and in the isolated epithelial cells derived from the mammary explants of the parallel culture. The differential response of epithelial and fat cells to hormones was observed in the study of several enzymes involved in polyamine biosynthesis in cultured mammary tissue.7,8,13 Determination of Polyamine Content The content of putrescine, spermidine, and spermine in mammary explants was quantitated with ninhydrin using an amino acid analyzer) 7 About 50-100 mg of explants were homogenized in 1 ml of 3% perchloric acid. After centrifugation, the residue was washed by another 1 ml of 3% perchloric acid, and the combined supernatant fractions were neutralized with 4 N KOH. The precipitate was removed by centrifugation, and the 13 T. 14 T. 15 R. 16T. 17 D. 81
Oka, J. W. Perry, and K. Kano, Biochem. Biophys. Res. Commun. 79, 979 (1977). Sakai, D. W. Lundgren, and T. Oka, J. Cell. Physiol. 95, 259 (1978). T. Schimke, J. Biol. Chem. 39, 3809 (1964). Oka and J. W. Perry, Nature (London) 250, 660 (1974). W. Lundgren, P. M. Farrell, and P. A. di Sant'Agnese, Proc. Soc. Exp. Biol. Med. 152, (1976).
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supernatant solution was lyophilized. The dried residue was dissolved in a potassium chloride-potassium citrate buffer (2.34 M K+), pH 5.64, and assayed for the amount of polyamine. ~4 This method is more sensitive than the high-voltage electrophoretic method used in earlier studies. 8 In cultured mammary tissue from virgin mice, insulin increases the content of putrescine and spermidine about threefold over the initial level during a 3-day culture, whereas the content of spermine remains relatively unchanged. ~4 In culture of mammary tissue from midpregnant mice, the synergistic action of insulin, cortisol, and prolactin increases the concentration of spermidine in mammary epithelium about threefold in 2 days. 8 The combination of insulin and prolactin or the combination of insulin and cortisol enhances the polyamine level to a small extent. 8 The concentration of spermine in mammary epithelium increased approximately 30% during 2 days, but no appreciable difference was found among various combinations of the three hormones. 8 Uptake of Polyamines Mouse mammary gland has been shown to possess a transport system for spermidine, spermine, and putrescine.lS In order to measure the uptake of polyamine, 10-20 mg of explants were incubated in Medium 199 with appropriately labeled polyamines. The amount of radioactive polyamine added to the culture was 1-2 /zCi/ml and 0.01 /zCi/ml for 3Hlabeled and for 14C-labeled polyamine, respectively. Tritium- or 14C-labeled polyamines (specific activity 100-1000 mCi/mmol and 5-20 mCi/mmol, respectively) were obtained from commercial sources, checked for purity, and, whenever necessary, purified by high-voltage electrophoresis prior to use. s After addition of labeled polyamine, explants were harvested at various times, weighed, and placed for washing on a Whatman GS/C disk filter paper that had been immersed for at least 15 rain in Medium 199 containing 40 mM corresponding unlabeled polyamine to prevent adsorption of the residual external radioactive polyamines. Explants were washed on a Millipore filter apparatus with 15 ml of Medium 199 and 4 ml of phosphate-buffered 0.15 M NaCI (pH 7.2), using suction. Each washing solution also contained the appropriate unlabeled polyamine at 40 mM. This washing procedure removed approximately 10% of the radioactivity associated with explants, which presumably represented nonspecific adsorption of the polyamines to the cells. The washed explants on the filters were transferred into a scintillation vial, digested with 1 ml of tissue solubilizer, and assayed for radioactivity is K. Kano and T. Oka, J. Biol. Chem. 251, 2795 (1976).
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in a toluene-based scintillation fluid with a liquid scintillation spectrometer. Insulin stimulates the uptake of polyamines by enhancing Vmax for polyamine influx and preventing efflux of polyamine. ~8 Prolactin, in the presence of insulin, elicits a greater increase in Vmaxfor polyamine uptake. 18 Assessment of the Function of Polyamines in the Mammary Gland Hormonal stimulation of the mammary gland in culture causes marked enhancement of polyamine accumulation prior to acceleration of DNA synthesis ~4and milk-protein synthesis. 8 Since the mammary epithelium in virgin mice is essentially nonproliferative, but can be induced to undergo DNA synthesis in culture with insulin, this system is suited for examining the role of polyamines in the proliferation of mammary epithelium. On the other hand, the mammary cells in pregnant mice are in an active phase of proliferation and can be readily stimulated to undergo differentiation. Thus organ culture of pregnant mouse mammary tissue provides a system to study the function of polyamines in mammary differentiation. The biological importance of polyamine accumulation in the development of the mammary gland can be assessed by employing various culture conditions in which the intracellular level of polyamines is varied by alterations in hormone combination, 8 osmolarity, 6 amino acid composition of culture medium, ~6use of several inhibitors of ornithine decarboxylase, 19 and S-adenosylmethionine decarboxylase, 8 and finally, by use of mammary explants derived from virgin and pregnant mice containing initially a low and a high level of polyamines.~4,~9 It is also possible to change the intracellular concentration of polyamines by exogenously adding them to culture. 8,~4 Combinations of these experimental approaches have yielded some useful information concerning the role of polyamines in the development of the mammary gland. In cultured mammary explants from virgin mice, both putrescine and spermidine appear to be important for hormonal induction of DNA synthesis in mammary epithelium. 14,~9Furthermore, several lines of evidence suggest that spermidine (0.04 raM) can simulate the action of glucocorticoid on induction of a-lactalbumin synthesis in cultured tissue from midpregnant mice. 8 It is noteworthy that stimulation of synthesis of milk protein in rabbit mammary tissue can occur in the presence of only insulin and prolactin and does not require the action of glucocorticoid and spermidine. 2° On the other hand, synthesis of milk protein in rat mammary 19 H. Inoue and T. Oka, J. Biol. Chem. 255, 3308 (1980). 20 L. M. Houdebine, E. Devinoy, and C. Delouis, Biochimie 60, 735 (1978).
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gland is dependent on both glucocorticoid and spermidine, but spermidine, in place of cortisol, is unable to act synergistically with insulin and prolactin. 21 The reason for the observed species differences remains unknown. It should be emphasized that some caution must be exercised in interpreting these experimental results obtained by use of enzyme inhibitors and supraphysiological conditions because of other possible side effects. 21 F. F. Bolander and Y. J. Topper, Biochem. Biophys. Res. Commun. 90, 1131 (1979).
[67] O r n i t h i n e D e c a r b o x y l a s e A s s a y P e r m i t t i n g E a r l y D e t e r m i n a t i o n o f H i s t o c o m p a t i b i l i t y in t h e M i x e d Lymphocyte Reaction By
ACHILLES
A.
D E M E T R I O U , CELIA W H I T E TABOR,
a n d HERBERT TABOR
The rejection of transplanted organs is a consequence of genetic disparity between donor and recipient. The human major histocompatibility complex (MHC), termed HLA, is localized on chromosome six and it includes three loci (A, B, C) whose products can be recognized by complement-dependent lymphocytotoxicity techniques. Another important component is the HLA-D locus, whose products are recognizable by the mixed lymphocyte culture (MLC) test. In the MLC test, lymphocytes from a donor and a recipient are mixed in vitro, and antigenic determinants coded by the HLA-D region, which are present on the cell surface, provoke a mixed lymphocyte reaction of proliferation and blast transformation when genetically dissimilar lymphocytes interact. At present, the most widely used assay of mixed lymphocyte reactivity measures the incorporation of tritiated thymidine into DNA and requires 4-5 days. 1 This long waiting period has hampered the application of this technique to the prospective selection of recipients for cadaver organ transplantation. Changes in the activity of ornithine decarboxylase, at 18 hr after mixing in the MLC, can be used as an early, sensitive indicator of the degree of histocompatibility.2 1 F. H. Bach and K. Hirschhorn, Science 142, 813 (1964). 2 A. A. Demetriou, M. W. Flye, C. W. Tabor, and H. Tabor, Transplantation 27, 190 (1979).
METHODS IN ENZYMOLOGY,VOL. 94
Copyright © 1983by Academic Press, inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9