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Effect of sodium butyrate on the granulopoiesis of mastocytoma cells
Copyright 0 197X by Academic Prew. Inc. All nghtb of reproduction in any form reaerwd wl4-4s27/79/oloolc-l~x$o?.~x~/o
Experimental Cell Research 118 (1979) 1.5-22
EFFECT
OF SODIUM
BUTYRATE
ON THE GRANULOPOIESIS
OF MASTOCYTOMA Y. MORI,’ H. AKEDO,Z K. TANAKA,’
CELLS
Y. TANIGAKI’
and M. OKADA
‘Depurtment of Cell Biology, 2Depnrrment of Biochemistry, 3Department of Electron Microscop?,. and the Centerfor Adult Diseases, Osaka, I-3-3, Nakamichi, Higashinari, Osaka, 537, Japan
SUMMARY The effect of sodium butyrate on mastocytoma p-815-4 cells was examined. In the presence of butyrate, the proliferation of mastocytoma p-815-4 cells decreased without any detectable cellular degeneration, and the average cell volume of the butyrate-treated cells increased. These cells tended to adhere to the substratum, and manifested their morphological changes. On the second day after the addition of butyrate. basophilic granules appeared around the Golgi area and gradually increased in number and in size. Ultrastructural examination with the electron microscope showed a well developed Golgi complex, numerous basophilic granules, many profiles of endoplasmic reticulum and many microvilli. These changes were maximized 34 days after the addition of this agent. The agglutination of the butyrate-treated cells by concanavalin A (ConA) or wheat germ agglutinin (WGA) did not change significantly. The induction of granulopoiesis of the cells in the presence of butyrate was prevented by the addition of actinomycin D or cycloheximide.
It has been reported that short-chain fatty acids, such as butyric and caproic acid, inhibit the proliferation of cells in tissue culture and alter their morphology by producing a drastic extension of cell processes [14]. Altenburg et al. reported that the morphology of virally transformed cells could be reversed to the normal by a striking elaboration of cytoplasmic microtubules and microfilaments by butyrate [7]. Leder & Leder reported that butyrate induced globin synthesis in Friend erythroleukemia cells [8]. Riggs et al. demonstrated that there was a reversible increase in acetylation of histone H4 of HeLa and Friend erythroleukemia cells in the presence of butyrate 191. This agent is also known to increase the specific activity of membranebound enzyme such as alkaline phosphatase, sialyltransferase or adenylate cyclase [lO-121. However, the mechanism of the ac2-781802
tion is still obscure. In the present report we describe striking morphological alterations of mastocytoma cells that could have resulted from a change in membrane biogenesis induced by butyrate.
MATERIALS
AND METHODS
Culture cells Mastocytoma p-815 cells transplanted and grown in ascites form in the peritoneal cavity of a DBA mouse, were collected and cultured in a milk bottle in suspension at 3PC in RPMI-1640 (Nissui Chem., Osaka) supplemented with 15% fetal calf serum (FCS) (Gibco, N.Y.). Mastocytoma p-81.5-4,p-815-6 and p-815-9 cells were cloned from the p-815 cells and maintained in suspension culture. The cell lines of p-815-4 and p815-6 lacked cytoplasmic granules, while p-815-9 cells had a few basophilic granules. The cell number in cultures was determined by a Coulter Counter ZBI (Coulter Electronics Inc, Hialeah, Fla). The population distribution as a function of cell volume was determined by using a cell volume plotter which was connected to this Coulter Counter. Polystyrene DVB SR-15 particles (0 13.5 pm; Particle Information Service Inc, Calif.) were used as a standard. Exp Cell Res 118 lIY7Y)
16
Mori et ~1.
cell vol. (X lo-” wm”); ~rdi/mte: cell no. -. Cells without butyrate; ---, cells with I mM butyrate for 3 days; ---. cells with 2 mM butyrate for 3 days.
Fig. 2. Abscissa:
time (days); ordinate: no. of cells/ml. Control cells; 0- - -0, cells with 1 mM butyrate; A---A, cells with 2 mM butyrate. Effect of butyrate on the growth of mastocytoma p-815-4 cells.
Fig. I. Abscissa:
a-0,
Cytology Mastocytoma cells were collected by centrifugation at 200 g for 5 min, and were smeared on a glass slide and stained with May-Giemsa, alcian blue (pH 2.9), toluidine blue (pH 2.5) and PAS. For electron microscopic examinations, the cells were centrifuged and the cell pellet was fixed with 2.5 % glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2), post-fixed with 1% osmium tetroxide, dehydrated with increasing concentrations of ethanol followed by propylene oxide, and finally embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate, and then examined with a JEM 100-b electron microscope (Nippon Denshi, Tokyo).
Assay for cell agglutination by lectins The procedure for testing cell agglutination by concanavalin A (ConA) and wheat germ agglutinin &VGA) was described previously [ 131.
Chemicals ConA was purified and crystallized as reported in a previous paper [14], and WGA was purified by the method described by Marchesi [15]. Sodium butyrate was purchased from Katayama Chem. (Osaka) and cycloheximide and actinomycin D from Sigma Chem. (St Louis).
RESULTS When mastocytoma p-815-4 cells were grown in the presence of 1 and 2 mM butyrate, the growth rate was decreased with inExp Cdl Res 118 (1979)
creasing concentrations of butyrate (fig. 1). The doubling time of cells was prolonged to 28.9 h in the presence of 1 mM butyrate and 41.9 h in 2 mM, in comparison with 22.2 h for untreated cells. The saturation density also decreased to 40% in the presence of I mM and 20% in 2 butyrate. Concentrations of butyrate below 0.2 mM had no effect on the cell growth. The viability of cells was unaffected at the concentrations of butyrate below 2 mM, when examined by the exclusion test with trypan blue. During suspenFig. 3. Morphological changes of mastocytoma p-815-4 cells in the presence of butyrate (light microscopy). (a) A cell in the absence of butyrate. It has prominent nucleoli and a basophilic cytoplasm; (b) a cell in the presence of 2 mM butyrate for 4 days. Note the large cell volume and many basophilic granules in the cytoplasm. Giemsa stain, x 1000. Fig. 4. Morphologicaj changes of mastocytoma p-815-4 cells in the presence of butyrate (phase contrast microscopy). (a) Cells in the absence of butyrate. They are of a round form and remain floating. (b) Cells in the presence of 2 mM butyrate for 24 h. They tend to adhere to the substratum and to show a spindle form. x too. Fig. 5. Electron micrographs of mastocytoma p-815-4 cells cultured in the presence and in the absence of butyrate. (a) A cell in the absence of butyrate. Many polysomes and a few small profiles of endoplasmic reticulum are seen. (b) A cell in the presence of 2 mM butyrate for 3 days. Note a well developed Golgi complex, many granules, the increased endoplasmic reticulum and microvilli. Bar, 2.5 pm.
Granulopoiesis by butyrate
17
3a
Exp Cd Re.\ II8 (1979)
plasm that was clearly orthochromatic with toluidine blue stain. Nuclei were round or slightly oval with uniformly dispersed chroDegree of agglutination matin and several prominent nucleoli. By Butyrate by ConA (in wg/ml) cont. ultrastructural examination with the elec0 I 5 10 50 100 (mM) tron microscope, the cells showed many 0 + + ++ polysomes in the cytoplasm with a poorly + + ++ 2 developed Golgi complex. A few small profiles of endoplasmic reticulum were obDegree of agglutination by WGA (in pg/ml) served (figs 3 a-5 (1). When mastocytoma p-815-4 cells were 0 I 5 IO 50 100 + ++ 0 cultured in the presence of butyrate, the + 2 ++ cellular morphology changed markedly as shown in figs 3 b-7 h . The shape of nuclei Mastocytoma p-815-4 cells were cultured with or without butvrate for 3 davs. Cells were washed with PBS changed to oval, elongated or kidney form, and resuspended in PBS at a concentration of SX 1O’j and occasionally it exhibited irregular outcells/ml, and the capabilitv of cell agglutination was examined after incubation-with lectinsat 37°C for 15 lines with deep indentations. The cytomin. The degree of agglutination was scored on a + + + plasm was less basophilic, and the nucleoto + scales: + + +, agglutinate composed of one large mass; ++, agglutinates composed of several masses; cytoplasmic ratio was smaller than that of f, formation of many small clumps: -. no agglutinauntreated cells. A number of basophilic tion granules were observed throughout the cytoplasm. These granules were stained sion culture in the presence of butyrate, mastocytoma cells tended to adhere to the blue with alcian blue stain, and they were substratum: at 24 h about half of the cells metachromatic with toluidine blue stain but adhered to the substratum and displayed were negative to PAS stain. Granulopoiesis cytoplasmic extension. These cells showed started around the Golgi area about 24 h a spindle form with a fine extension at one after the addition of butyrate, and at this or both ends of the cell, whereas in the stage the granules were stained fine purple absence of butyrate the cells maintained a red with Giemsa stain. At 48 h, these granround form and remained floating in the me- ules gradually increased in number and in size and were stained purple blue with dium. Fig. 2 shows the effect of butyrate on the Giemsa stain. The maximal extent of granpopulation distribution as a function of cell ulopoiesis was seen on the 4th day after the volume. The mean, modal and medium cell addition of butyrate and it was maintained volumes of untreated cells were 807, 650 at least for 70 days (11 passages). Ultraand 775 pm3. The values of the treated cells structural examination with the electron miwere increased to 1089, 725 and 975 pm” croscope showed that the cells possessed in the presence of 1 mM butyrate, and to many microvilli on the cell surface in con1321, 1000 and 1250 pm3 in 2 mM butyrate. trast to the untreated cells which had few Figs 3-7 show the morphology of masto- microvilli. The well developed Golgi comcytoma p-815-4 cells cultured in the pres- plex was located in the close vicinity of the ence or in the absence of butyrate. Masto- nucleus, and was comprised of cysternae cytoma p-815-4 cells in the absence of bu- and many small vesicles. Many small vactyrate were round with a basophilic cyto- uoles were observed along the cysternae, Erp Cell Rr., II8 (1979)
Granulopoiesis by butyrate
Fig. 6. The Golgi area of mastocvtoma o-815-4 cells
c&ured in the presence of 2 mM butyratk for 3 days. The well developed Golgi complex consists of Golgi cystemae and vacuoles. In the center of the Golgi co;plex, centrioles are observed. Many microtubules are seen to emerge from the centrioles. Many granules
suggesting that they would be derived from Golgi vesicles. Centrioles with a characteristic fine tubular structure were seen in the center of Golgi complex, and many microtubules were seen to emerge from the centrioles. The rough surfaced endoplasmic reticulum increased throughout the cytoplasm, and some of them were enlarged. Many granules of varying structures from 0.1 to 1.2 pm in size appeared in the cytoplasm, especially around the Golgi com-
19
(type 1, 2 and 4) are observed as if they have been elaborated from the Golgi complex. Fig. 7. The various basophilic granules of mastocytoma p-815-4 cells induced by butyrate. (a, b) Type 1; (c) type 2; (d) type 3; (e) type 4.
plex. They were mostly surrounded by a limiting membrane. These granules can be subtyped as follows: the common type of granules (0.1-0.6 pm, mostly less than 0.5 pm in diameter) consisted of finely granular or coarse reticular material with a moderate density (type 1). The second type of granules (0 OA-O.9 pm) contained homogeneous or amorphous material of varying electron densities and occasionally contained the scroll-like structures, such as Exp Celi
Res
i 18 ( 1979)
20
Mori
et (11
those described by Brinkman [16]. In the third type (0 0.40.7 Frn) a parallel crystalloid pattern was seen. The fourth type (0 0.4-I .2 pm) was an aggregate of the first and second types with a limiting membrane which was partly absent in some cases. The granules seemed to originate from vacuoles around the Golgi cysternae, because the larger vacuoles showed a granular or reticular content although the smaller vacuoles showed a flocculent moderate density. The replacement of culture medium with the one lacking butyrate resulted in the reversion of cell morphology which was characterized by disappearance of the granules after 3-4 days of culture. The cell growth also recovered to the normal rate. One of the other cloned cell lines, p-815 9, which had originally a few basophilic granules, showed a similar increase in granulopoiesis in the presence of butyrate. However, the cloned cell line, p-815-6, did not show any granulation, while the other morphological changes were induced by this agent. The difference in inducibility of granulopoiesis suggests that there were genetic differences between these clones. In order to examine any modification of the cell surface by treatment with butyrate, the cell agglutination by ConA and by WGA was investigated. As shown in table 1, there was no difference in the agglutinability by ConA and by WGA between the cells cultured with or without butyrate. The granulopoiesis in mastocytoma cells induced by butyrate was inhibited by the addition of 0.1 pg/ml actinomycin D or 1X lop5 M cycloheximide to the culture medium. DISCUSSION Mastocytoma p-815 cells were originally developed by Dunn & Potter, who reported
that the cells contained many granules stained metachromatically with toluidine blue [17]. The morphological details of p815 cells were later reported by Bloom [ 181. The cells available at present are less granulopoietic. We have carried out cloning of cells from mastocytoma p-815 cells. A progeny of each cloned cell has a characteristic granularity: p-815-4 and p-815-6 cells are clearly orthochromatic with toluidine blue stain, whereas p-815-9 cells have a few basophilic granules. The present study demonstrates that the addition of butyrate to the culture of mastocytoma p-815-4 cells results in a marked development of the Golgi complex, from which a number of basophilic granules are formed. These granules are stained blue by alcian blue and metachromatic with toluidine blue stain. Ultrastructural examination with the electron microscope shows that butyrate-treated cells have granules characteristic of tissue mast cells [16, 19-221: the granules contain granular, reticular or crystalloid structures, with which the scroll-like structures are often seen in the some granules. However, most granules do not seem to be in a complete maturation, because they show a granular or reticular structure (type I), and seem to remain in the early stage of normal development of granules of tissue mast cells. Only a few granules (type 3) contain a crystalloid structure. Moreover, most granules are smaller than those of tissue mast cells and locate around the Golgi complex, while those of tissue mast cells are scattered throughout the cytoplasm. Combs reported the granulopoiesis of mast cells in the skin of rat embryos [23]. He described that the synthesis of granules began with the formation of small immature granules (progranules) in the center of the Golgi area. The progranules fused and
Grunulopoirsis
by butyrrrtr
21
fine granular materials accumulated in the tigated the effect of butyrate on the memfused granules. The resultant dense and fine brane structure and property which affect granular components then appeared to be cell agglutination. reorganized in a crystalloid structure. Terry In agreement with other investigators [5, et al. reported that the granulopoiesis of 71, the morphological changes induced by basophilic leukocytes in the bone marrow butyrate, especially the granulopoiesis, are occurred during cell differentiation [24]. inhibited by the simultaneous addition of They demonstrated that the granules elabo- actinomycin D or cycloheximide. This sugrated from Golgi vacuoles increased in their gests that butyrate acts on the level of transize, and finally developed into matured scription of genes. Recently Riggs et al. found in HeLa and Friend erythroleukemia granules which contained a crystalloid structure. The granulopoiesis in masto- cells that the acetylation of histone H4 was cytoma cells induced by butyrate is very greatly increased when the cells were similar to that in the process of differentia- treated with butyrate [9]. However, the pretion of normal tissue mast cells and baso- cise mechanism by which butyrate-induced philic leukocytes. The cytoplasmic gran- granulopoiesis with concomitant changes of ules, long protoplasmic processes, oval or the other cellular phenotype is unclear and kidney-shaped nuclei and numerous micro- further investigation is needed. villi are outstanding features of tissue mast cells. Mastocytoma cells grown in the presence of butyrate seem to acquire many such REFERENCES characteristic of tissue mast cells, though Wright, J A, Exp cell res 78 (1973) 456. the conversion to normal morphology is inGinsburg, E, Salomon, D, Sreevalsam, T & Freese. E. Proc natl acad sci US 70 (1973) 2457. complete. Ghosh; N K, Deutsch, S I, Griffin, M’J & Cox, R ConA or WGA, which binds to carboP, J cell physio186 (1975) 663. Griffin, M J, Price, G H, Bazzel, K L, Cox, R P & hydrate residues on the cell surface is Ghosh. N K. Arch biochem biophvs _ _ 164 (1974) known to agglutinate malignant trans619. 5. Henneberry, R C & Fishman, P H, Exp cell res formed cells much more extensively than 103(1976) 55. normal, untransformed cells [2.5, 261. The 6. Storrie, B, Puck, T T & Wenger, L, J cell physiol 94 (1978) 69. cell agglutination by these lectins is also re7. Altenburg, B C, Via, D P & Steiner, S H, Exp cell ported to change in the course of cell difres 102(1976) 223. ferentiation in embryonic development [ 13, 8. Leder, A & Leder, P, Cell 5 (1975) 319. 9. Riggs, M G, Whittaker, R G, Neumann, J R & 271, or in the revertant cells from malignant Ingram, V M, Nature 268 (1977) 462. transformed cells [28]. The occurrence of 10. Kovama. H & Ono. T. J cell uhvsiol 88 (1976) 49. 11. Simmons, J L, Fishman, P H; Freese, E‘& Brady, agglutination in any given cell system is due R 0, J cell bio166 (1975)414. to several interrelated factors: the number 12. Fishman, P H, Bradley, R M & Henneberry, R C, Arch biochem biophys 172 (1976) 618. of receptors in the membrane, the mobility 13. Mori, Y, Akedo,-H; Tanigaki, Y & Okada, M, Cytobiol I2 (1976) 397. of the lectin-binding sites, cell charge re14. Akedo, H, Mori, Y, Tanigaki, Y, Shinkai, K & pulsive forces, cell surface rigidity and/or Morita, K, Biochim biophys. acta 271 (1972) 378. membrane-associated components in cyto- 15. Marchesi. V T. Methods in enzvmoloev (ed V Ginsburg) 1st edn, vol. 28, part 2, p. ?54.~Acaplasm which link with cell surface strucdemic Press, New York (1972). tures. In this experiment, no difference was 16. Brinkman, G L, J ultrastruct res 23 (1%8) 115. 17. Dunn, T B & Potter, M J, J natl cancer inst I8 observed between cells cultured with and (1957) 587. without butyrate. We have not yet inves- 18. Bloom, G D, Ann NY acad sci 103(1968) 1I5 EA, Cell Res I I8 (1979)
22
Mori
et 01.
19. Weinstock, A B Albright, J T. J ultrastruct res 17 (1967) 245. 20. Taichman, N S, J ultrastruct res 32 (1970) 284. 21. Barnett, M L, J ultrastruct res 43 (1973) 247. 22. Matsuura, S & Tashiro. Y, Recent progress in electron microscopy of cell and tissue (ed E Yamada, V Mizuhira. K Kurosumi & T Nagano) 1st edn, p. 69. lgaku Shoin, Tokyo (1976). 23. Combs, J W, J cell biol 31 (1966) 563. 24. Terry, R W, Bainton, D F & Farquhar, M G. Lab invest 21 (1969) 65. 25. Inbar, M & Sachs. L. Proc natl acad sci US 63 (1969) 1418.
26. Burger, M M & Goldberg, A R. Proc natl acad \CI US 57 (1967) 3.59. 27. Kleinschuster. S J & Moscona. A A. Esp cell r-e\ 70 (1972) 397. 28. Gulp, L A. Grimes, W J &Black, P H. J cell biol 50 (1971)682.
Received March 29, 1978 Revised version received August 2 I. 1978 Accepted August 23. 1978
Experimental Cell Research 118 (1979) 1.5-22
EFFECT
OF SODIUM
BUTYRATE
ON THE GRANULOPOIESIS
OF MASTOCYTOMA Y. MORI,’ H. AKEDO,Z K. TANAKA,’
CELLS
Y. TANIGAKI’
and M. OKADA
‘Depurtment of Cell Biology, 2Depnrrment of Biochemistry, 3Department of Electron Microscop?,. and the Centerfor Adult Diseases, Osaka, I-3-3, Nakamichi, Higashinari, Osaka, 537, Japan
SUMMARY The effect of sodium butyrate on mastocytoma p-815-4 cells was examined. In the presence of butyrate, the proliferation of mastocytoma p-815-4 cells decreased without any detectable cellular degeneration, and the average cell volume of the butyrate-treated cells increased. These cells tended to adhere to the substratum, and manifested their morphological changes. On the second day after the addition of butyrate. basophilic granules appeared around the Golgi area and gradually increased in number and in size. Ultrastructural examination with the electron microscope showed a well developed Golgi complex, numerous basophilic granules, many profiles of endoplasmic reticulum and many microvilli. These changes were maximized 34 days after the addition of this agent. The agglutination of the butyrate-treated cells by concanavalin A (ConA) or wheat germ agglutinin (WGA) did not change significantly. The induction of granulopoiesis of the cells in the presence of butyrate was prevented by the addition of actinomycin D or cycloheximide.
It has been reported that short-chain fatty acids, such as butyric and caproic acid, inhibit the proliferation of cells in tissue culture and alter their morphology by producing a drastic extension of cell processes [14]. Altenburg et al. reported that the morphology of virally transformed cells could be reversed to the normal by a striking elaboration of cytoplasmic microtubules and microfilaments by butyrate [7]. Leder & Leder reported that butyrate induced globin synthesis in Friend erythroleukemia cells [8]. Riggs et al. demonstrated that there was a reversible increase in acetylation of histone H4 of HeLa and Friend erythroleukemia cells in the presence of butyrate 191. This agent is also known to increase the specific activity of membranebound enzyme such as alkaline phosphatase, sialyltransferase or adenylate cyclase [lO-121. However, the mechanism of the ac2-781802
tion is still obscure. In the present report we describe striking morphological alterations of mastocytoma cells that could have resulted from a change in membrane biogenesis induced by butyrate.
MATERIALS
AND METHODS
Culture cells Mastocytoma p-815 cells transplanted and grown in ascites form in the peritoneal cavity of a DBA mouse, were collected and cultured in a milk bottle in suspension at 3PC in RPMI-1640 (Nissui Chem., Osaka) supplemented with 15% fetal calf serum (FCS) (Gibco, N.Y.). Mastocytoma p-81.5-4,p-815-6 and p-815-9 cells were cloned from the p-815 cells and maintained in suspension culture. The cell lines of p-815-4 and p815-6 lacked cytoplasmic granules, while p-815-9 cells had a few basophilic granules. The cell number in cultures was determined by a Coulter Counter ZBI (Coulter Electronics Inc, Hialeah, Fla). The population distribution as a function of cell volume was determined by using a cell volume plotter which was connected to this Coulter Counter. Polystyrene DVB SR-15 particles (0 13.5 pm; Particle Information Service Inc, Calif.) were used as a standard. Exp Cell Res 118 lIY7Y)
16
Mori et ~1.
cell vol. (X lo-” wm”); ~rdi/mte: cell no. -. Cells without butyrate; ---, cells with I mM butyrate for 3 days; ---. cells with 2 mM butyrate for 3 days.
Fig. 2. Abscissa:
time (days); ordinate: no. of cells/ml. Control cells; 0- - -0, cells with 1 mM butyrate; A---A, cells with 2 mM butyrate. Effect of butyrate on the growth of mastocytoma p-815-4 cells.
Fig. I. Abscissa:
a-0,
Cytology Mastocytoma cells were collected by centrifugation at 200 g for 5 min, and were smeared on a glass slide and stained with May-Giemsa, alcian blue (pH 2.9), toluidine blue (pH 2.5) and PAS. For electron microscopic examinations, the cells were centrifuged and the cell pellet was fixed with 2.5 % glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2), post-fixed with 1% osmium tetroxide, dehydrated with increasing concentrations of ethanol followed by propylene oxide, and finally embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate, and then examined with a JEM 100-b electron microscope (Nippon Denshi, Tokyo).
Assay for cell agglutination by lectins The procedure for testing cell agglutination by concanavalin A (ConA) and wheat germ agglutinin &VGA) was described previously [ 131.
Chemicals ConA was purified and crystallized as reported in a previous paper [14], and WGA was purified by the method described by Marchesi [15]. Sodium butyrate was purchased from Katayama Chem. (Osaka) and cycloheximide and actinomycin D from Sigma Chem. (St Louis).
RESULTS When mastocytoma p-815-4 cells were grown in the presence of 1 and 2 mM butyrate, the growth rate was decreased with inExp Cdl Res 118 (1979)
creasing concentrations of butyrate (fig. 1). The doubling time of cells was prolonged to 28.9 h in the presence of 1 mM butyrate and 41.9 h in 2 mM, in comparison with 22.2 h for untreated cells. The saturation density also decreased to 40% in the presence of I mM and 20% in 2 butyrate. Concentrations of butyrate below 0.2 mM had no effect on the cell growth. The viability of cells was unaffected at the concentrations of butyrate below 2 mM, when examined by the exclusion test with trypan blue. During suspenFig. 3. Morphological changes of mastocytoma p-815-4 cells in the presence of butyrate (light microscopy). (a) A cell in the absence of butyrate. It has prominent nucleoli and a basophilic cytoplasm; (b) a cell in the presence of 2 mM butyrate for 4 days. Note the large cell volume and many basophilic granules in the cytoplasm. Giemsa stain, x 1000. Fig. 4. Morphologicaj changes of mastocytoma p-815-4 cells in the presence of butyrate (phase contrast microscopy). (a) Cells in the absence of butyrate. They are of a round form and remain floating. (b) Cells in the presence of 2 mM butyrate for 24 h. They tend to adhere to the substratum and to show a spindle form. x too. Fig. 5. Electron micrographs of mastocytoma p-815-4 cells cultured in the presence and in the absence of butyrate. (a) A cell in the absence of butyrate. Many polysomes and a few small profiles of endoplasmic reticulum are seen. (b) A cell in the presence of 2 mM butyrate for 3 days. Note a well developed Golgi complex, many granules, the increased endoplasmic reticulum and microvilli. Bar, 2.5 pm.
Granulopoiesis by butyrate
17
3a
Exp Cd Re.\ II8 (1979)
plasm that was clearly orthochromatic with toluidine blue stain. Nuclei were round or slightly oval with uniformly dispersed chroDegree of agglutination matin and several prominent nucleoli. By Butyrate by ConA (in wg/ml) cont. ultrastructural examination with the elec0 I 5 10 50 100 (mM) tron microscope, the cells showed many 0 + + ++ polysomes in the cytoplasm with a poorly + + ++ 2 developed Golgi complex. A few small profiles of endoplasmic reticulum were obDegree of agglutination by WGA (in pg/ml) served (figs 3 a-5 (1). When mastocytoma p-815-4 cells were 0 I 5 IO 50 100 + ++ 0 cultured in the presence of butyrate, the + 2 ++ cellular morphology changed markedly as shown in figs 3 b-7 h . The shape of nuclei Mastocytoma p-815-4 cells were cultured with or without butvrate for 3 davs. Cells were washed with PBS changed to oval, elongated or kidney form, and resuspended in PBS at a concentration of SX 1O’j and occasionally it exhibited irregular outcells/ml, and the capabilitv of cell agglutination was examined after incubation-with lectinsat 37°C for 15 lines with deep indentations. The cytomin. The degree of agglutination was scored on a + + + plasm was less basophilic, and the nucleoto + scales: + + +, agglutinate composed of one large mass; ++, agglutinates composed of several masses; cytoplasmic ratio was smaller than that of f, formation of many small clumps: -. no agglutinauntreated cells. A number of basophilic tion granules were observed throughout the cytoplasm. These granules were stained sion culture in the presence of butyrate, mastocytoma cells tended to adhere to the blue with alcian blue stain, and they were substratum: at 24 h about half of the cells metachromatic with toluidine blue stain but adhered to the substratum and displayed were negative to PAS stain. Granulopoiesis cytoplasmic extension. These cells showed started around the Golgi area about 24 h a spindle form with a fine extension at one after the addition of butyrate, and at this or both ends of the cell, whereas in the stage the granules were stained fine purple absence of butyrate the cells maintained a red with Giemsa stain. At 48 h, these granround form and remained floating in the me- ules gradually increased in number and in size and were stained purple blue with dium. Fig. 2 shows the effect of butyrate on the Giemsa stain. The maximal extent of granpopulation distribution as a function of cell ulopoiesis was seen on the 4th day after the volume. The mean, modal and medium cell addition of butyrate and it was maintained volumes of untreated cells were 807, 650 at least for 70 days (11 passages). Ultraand 775 pm3. The values of the treated cells structural examination with the electron miwere increased to 1089, 725 and 975 pm” croscope showed that the cells possessed in the presence of 1 mM butyrate, and to many microvilli on the cell surface in con1321, 1000 and 1250 pm3 in 2 mM butyrate. trast to the untreated cells which had few Figs 3-7 show the morphology of masto- microvilli. The well developed Golgi comcytoma p-815-4 cells cultured in the pres- plex was located in the close vicinity of the ence or in the absence of butyrate. Masto- nucleus, and was comprised of cysternae cytoma p-815-4 cells in the absence of bu- and many small vesicles. Many small vactyrate were round with a basophilic cyto- uoles were observed along the cysternae, Erp Cell Rr., II8 (1979)
Granulopoiesis by butyrate
Fig. 6. The Golgi area of mastocvtoma o-815-4 cells
c&ured in the presence of 2 mM butyratk for 3 days. The well developed Golgi complex consists of Golgi cystemae and vacuoles. In the center of the Golgi co;plex, centrioles are observed. Many microtubules are seen to emerge from the centrioles. Many granules
suggesting that they would be derived from Golgi vesicles. Centrioles with a characteristic fine tubular structure were seen in the center of Golgi complex, and many microtubules were seen to emerge from the centrioles. The rough surfaced endoplasmic reticulum increased throughout the cytoplasm, and some of them were enlarged. Many granules of varying structures from 0.1 to 1.2 pm in size appeared in the cytoplasm, especially around the Golgi com-
19
(type 1, 2 and 4) are observed as if they have been elaborated from the Golgi complex. Fig. 7. The various basophilic granules of mastocytoma p-815-4 cells induced by butyrate. (a, b) Type 1; (c) type 2; (d) type 3; (e) type 4.
plex. They were mostly surrounded by a limiting membrane. These granules can be subtyped as follows: the common type of granules (0.1-0.6 pm, mostly less than 0.5 pm in diameter) consisted of finely granular or coarse reticular material with a moderate density (type 1). The second type of granules (0 OA-O.9 pm) contained homogeneous or amorphous material of varying electron densities and occasionally contained the scroll-like structures, such as Exp Celi
Res
i 18 ( 1979)
20
Mori
et (11
those described by Brinkman [16]. In the third type (0 0.40.7 Frn) a parallel crystalloid pattern was seen. The fourth type (0 0.4-I .2 pm) was an aggregate of the first and second types with a limiting membrane which was partly absent in some cases. The granules seemed to originate from vacuoles around the Golgi cysternae, because the larger vacuoles showed a granular or reticular content although the smaller vacuoles showed a flocculent moderate density. The replacement of culture medium with the one lacking butyrate resulted in the reversion of cell morphology which was characterized by disappearance of the granules after 3-4 days of culture. The cell growth also recovered to the normal rate. One of the other cloned cell lines, p-815 9, which had originally a few basophilic granules, showed a similar increase in granulopoiesis in the presence of butyrate. However, the cloned cell line, p-815-6, did not show any granulation, while the other morphological changes were induced by this agent. The difference in inducibility of granulopoiesis suggests that there were genetic differences between these clones. In order to examine any modification of the cell surface by treatment with butyrate, the cell agglutination by ConA and by WGA was investigated. As shown in table 1, there was no difference in the agglutinability by ConA and by WGA between the cells cultured with or without butyrate. The granulopoiesis in mastocytoma cells induced by butyrate was inhibited by the addition of 0.1 pg/ml actinomycin D or 1X lop5 M cycloheximide to the culture medium. DISCUSSION Mastocytoma p-815 cells were originally developed by Dunn & Potter, who reported
that the cells contained many granules stained metachromatically with toluidine blue [17]. The morphological details of p815 cells were later reported by Bloom [ 181. The cells available at present are less granulopoietic. We have carried out cloning of cells from mastocytoma p-815 cells. A progeny of each cloned cell has a characteristic granularity: p-815-4 and p-815-6 cells are clearly orthochromatic with toluidine blue stain, whereas p-815-9 cells have a few basophilic granules. The present study demonstrates that the addition of butyrate to the culture of mastocytoma p-815-4 cells results in a marked development of the Golgi complex, from which a number of basophilic granules are formed. These granules are stained blue by alcian blue and metachromatic with toluidine blue stain. Ultrastructural examination with the electron microscope shows that butyrate-treated cells have granules characteristic of tissue mast cells [16, 19-221: the granules contain granular, reticular or crystalloid structures, with which the scroll-like structures are often seen in the some granules. However, most granules do not seem to be in a complete maturation, because they show a granular or reticular structure (type I), and seem to remain in the early stage of normal development of granules of tissue mast cells. Only a few granules (type 3) contain a crystalloid structure. Moreover, most granules are smaller than those of tissue mast cells and locate around the Golgi complex, while those of tissue mast cells are scattered throughout the cytoplasm. Combs reported the granulopoiesis of mast cells in the skin of rat embryos [23]. He described that the synthesis of granules began with the formation of small immature granules (progranules) in the center of the Golgi area. The progranules fused and
Grunulopoirsis
by butyrrrtr
21
fine granular materials accumulated in the tigated the effect of butyrate on the memfused granules. The resultant dense and fine brane structure and property which affect granular components then appeared to be cell agglutination. reorganized in a crystalloid structure. Terry In agreement with other investigators [5, et al. reported that the granulopoiesis of 71, the morphological changes induced by basophilic leukocytes in the bone marrow butyrate, especially the granulopoiesis, are occurred during cell differentiation [24]. inhibited by the simultaneous addition of They demonstrated that the granules elabo- actinomycin D or cycloheximide. This sugrated from Golgi vacuoles increased in their gests that butyrate acts on the level of transize, and finally developed into matured scription of genes. Recently Riggs et al. found in HeLa and Friend erythroleukemia granules which contained a crystalloid structure. The granulopoiesis in masto- cells that the acetylation of histone H4 was cytoma cells induced by butyrate is very greatly increased when the cells were similar to that in the process of differentia- treated with butyrate [9]. However, the pretion of normal tissue mast cells and baso- cise mechanism by which butyrate-induced philic leukocytes. The cytoplasmic gran- granulopoiesis with concomitant changes of ules, long protoplasmic processes, oval or the other cellular phenotype is unclear and kidney-shaped nuclei and numerous micro- further investigation is needed. villi are outstanding features of tissue mast cells. Mastocytoma cells grown in the presence of butyrate seem to acquire many such REFERENCES characteristic of tissue mast cells, though Wright, J A, Exp cell res 78 (1973) 456. the conversion to normal morphology is inGinsburg, E, Salomon, D, Sreevalsam, T & Freese. E. Proc natl acad sci US 70 (1973) 2457. complete. Ghosh; N K, Deutsch, S I, Griffin, M’J & Cox, R ConA or WGA, which binds to carboP, J cell physio186 (1975) 663. Griffin, M J, Price, G H, Bazzel, K L, Cox, R P & hydrate residues on the cell surface is Ghosh. N K. Arch biochem biophvs _ _ 164 (1974) known to agglutinate malignant trans619. 5. Henneberry, R C & Fishman, P H, Exp cell res formed cells much more extensively than 103(1976) 55. normal, untransformed cells [2.5, 261. The 6. Storrie, B, Puck, T T & Wenger, L, J cell physiol 94 (1978) 69. cell agglutination by these lectins is also re7. Altenburg, B C, Via, D P & Steiner, S H, Exp cell ported to change in the course of cell difres 102(1976) 223. ferentiation in embryonic development [ 13, 8. Leder, A & Leder, P, Cell 5 (1975) 319. 9. Riggs, M G, Whittaker, R G, Neumann, J R & 271, or in the revertant cells from malignant Ingram, V M, Nature 268 (1977) 462. transformed cells [28]. The occurrence of 10. Kovama. H & Ono. T. J cell uhvsiol 88 (1976) 49. 11. Simmons, J L, Fishman, P H; Freese, E‘& Brady, agglutination in any given cell system is due R 0, J cell bio166 (1975)414. to several interrelated factors: the number 12. Fishman, P H, Bradley, R M & Henneberry, R C, Arch biochem biophys 172 (1976) 618. of receptors in the membrane, the mobility 13. Mori, Y, Akedo,-H; Tanigaki, Y & Okada, M, Cytobiol I2 (1976) 397. of the lectin-binding sites, cell charge re14. Akedo, H, Mori, Y, Tanigaki, Y, Shinkai, K & pulsive forces, cell surface rigidity and/or Morita, K, Biochim biophys. acta 271 (1972) 378. membrane-associated components in cyto- 15. Marchesi. V T. Methods in enzvmoloev (ed V Ginsburg) 1st edn, vol. 28, part 2, p. ?54.~Acaplasm which link with cell surface strucdemic Press, New York (1972). tures. In this experiment, no difference was 16. Brinkman, G L, J ultrastruct res 23 (1%8) 115. 17. Dunn, T B & Potter, M J, J natl cancer inst I8 observed between cells cultured with and (1957) 587. without butyrate. We have not yet inves- 18. Bloom, G D, Ann NY acad sci 103(1968) 1I5 EA, Cell Res I I8 (1979)
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19. Weinstock, A B Albright, J T. J ultrastruct res 17 (1967) 245. 20. Taichman, N S, J ultrastruct res 32 (1970) 284. 21. Barnett, M L, J ultrastruct res 43 (1973) 247. 22. Matsuura, S & Tashiro. Y, Recent progress in electron microscopy of cell and tissue (ed E Yamada, V Mizuhira. K Kurosumi & T Nagano) 1st edn, p. 69. lgaku Shoin, Tokyo (1976). 23. Combs, J W, J cell biol 31 (1966) 563. 24. Terry, R W, Bainton, D F & Farquhar, M G. Lab invest 21 (1969) 65. 25. Inbar, M & Sachs. L. Proc natl acad sci US 63 (1969) 1418.
26. Burger, M M & Goldberg, A R. Proc natl acad \CI US 57 (1967) 3.59. 27. Kleinschuster. S J & Moscona. A A. Esp cell r-e\ 70 (1972) 397. 28. Gulp, L A. Grimes, W J &Black, P H. J cell biol 50 (1971)682.
Received March 29, 1978 Revised version received August 2 I. 1978 Accepted August 23. 1978