Inhibition of Melanosome Transformation in Embryonic Chick Pigment Retina Cultured In Vitro YOSHIKO K. TAKEUCHI, IKUO K . TAKEUCHI AND TAKA0 KAJISHIMA Biological Institute, Faculty of Science, Nagoya University, Nagoya 464, Japan
ABSTRACT
When the retinal pigment epithelial cells of the chick embryo are transferred to monolayer cultures, they lose their phenotypic trait melanin granules - after a few days. Within the first 24 hours almost all of the melanosomes and premelanosomes are transformed into the degradative structures of the dense bodies or the melanosome complexes. Then, within a few days, these structures disappear completely from the cytoplasm. Actinomycin D, added to the culture medium during the first four hours, almost completely prevents the transformation of melanosomes and premelanosomes. The inhibition of cell proliferation, caused by the addition of colcemid, does not prevent the transformation, though the time of initiation of transformation is delayed considerably. The mechanisms of the transformation of pigment granules are discussed
Retinal pigment cells of the chick embryo lose their phenotypic trait - melanin granules - under conditions of monolayer cell culture (Whittaker, ’63, ’67; Cahn and Cahn, ’66). This fact has been widely known as one of the phenomena of “dedifferentiation,” or, according to Weiss (‘49), “modulation of cell differentiation.” Whittaker (’63, ’67) has investigated the chemical characteristics of the melanin synthesizing systems of retinal pigment cells during depigmentation and found that declines in melanin concentration, tyrosinase activity, and rate of tyrosinase-dependent incorporation of tyrosine are initiated during the f m t few hours of culture. From these results, he has proposed the hypothesis that the cessation of tyrosinase synthesis in the pigment cells and the dilution of pigment granules by cell proliferation are the principal factors contributing to the loss of phenotype. To clarify the mechanisms of regulation of enzyme synthesis, Whittaker ( 68) mvestigated DNA, RNA, and protein synthesis in cultured retinal pigment cells by the method of determining the incorporation rate of labeled precursors into the cells; he found that a new DNA synthesis is not correlated with a loss of tyrosinase activity, whereas a close correlation does exist between a loss of tyrosinase synthesis J. EXP.ZOOL., 192: 391400.
and a rapid increase in the rate of both RNA and protein synthesis. He calculated the methylation ratio (methyl labevuridine label) of ribonuclease-digested RNA and concluded that the rapidly-synthesized RNAs in early cultures involve messenger RNA in addition to ribosomal RNA and transfer RNA. Believing that this messenger RNA might be used for the protein synthesis required for cell proliferation, he proposed the hypothesis of socalled “translational competition” between de novo synthesized messenger RNA and the preexisting tyrosinase messenger RNA. In recent electron microscopy studies we have demonstrated that the primary morphological change of this depigmentation is the transformation of pigment granules (Takeuchi et al., ’73). Within 24 hours after transferring the cells into culture, numerous electron-dense bodies (simply designated “dense bodies”) and melanosome complexes (probably formed by fusion of dense bodies with slowly degradating pigment granules) appeared in the cytoplasm. Conversion of pigment granules into dense bodies and melanosome complexes was followed by ultrastructural observation and by electron microscopic autora1 Present address: Department of Embryology, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan.
391
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INHIBITION OF MELANOSOME TRANSFORMATION
diography of 3H-Dopa incorporation. These results are in perfect accord with the depigmentation process of in vitro cultured mammalian melanoma cells (Mishima, '67; Novikoff et al., '68). However, what is involved in initiating the transformation of pigment granules in these cells remains unknown. In the present study, we used electron microscopy to examine the effects of actinomycin D and colcemid on the transformation of pigment granules. We hoped thereby to demonstrate the principal factor(s) required for initiating the depigmentation of retinal pigment cells in vitro. The nomenclature suggested by Fitzpatrick et al. ('66) for the terminology of pigment granules is used with slight modifications in this paper. MATERIALS AND METHODS
Cell culture Samples of retinal pigment epighelium from 6-day White Leghorn chick embryos (Hamburger-Hamilton's stage 28 [Hamburger and Hamilton, '511) were cut into pieces smaller than 1 X 1 mm, inoculated onto plasma clots (chick embryonic extract; chicken plasma [Difco], l : l ) , and grown as monolayer sheets. The culture medium consisted of equal parts of Tyrode's solution, horse serum, and 9-day chick embryonic extract. Penicilin G and dihydrostreptomycin were added to this medium. The methods of preparation and culture used in the present study were the same as those previously described (Takeuchi et al., '73). Treatments Actinomycin D (Merck) was added to the culture medium at a final concentration of 0.2 pglml. Colcemid (K & K Laboratories, Inc.) was used at a concentration of 2 X M. Fig. 1 Intact retinal pigment cell of a 6-day chick embryo. Numerous pigment granules in various developmental stages are observed. M, melanosome; Mt, mitochondria; N, nucleus; PM, premelanosome. X 22,800. Fig. 2 Twenty-four-hour cultured cells. 2a: Numerous dense bodies (DB) are observed in the cytoplasm. Nucleolus (No) becomes prominent. M, melanosome; N , nucleus. X 15,500. 2b: Melanosome complex (MC) contains many pigment granules. x 16,000.
393
Electron microscopy The cells were collected with a rubber policeman and fixed with Dalton's chromeosmium fixative (Dalton, '55) for two hours at 4°C. Dehydration was performed with an ethanol series and propylene oxide, then the specimens were embedded in Epon 812 according to Coulter ('67). Thin sections were cut on a Porter-Blum MT-2 ultramicrotome, double-stained with uranyl acetate and lead nitrate, and examined with a Hitachi HU 11-DS electron microscope at 75 kV. To obtain the definitive evidence, pigment granules were classified into four types - as premelanosome, melanosome (including the melanin granule described by Fitzpatrick et al., '66), dense body, and melanosome complex, and the pigment granules present in each cell were counted according to their respective types. The counts were performed on approximately 150 cells at each experimental stage. RESULTS
Control Intact retinal pigment cells from 6-day chick embryos contained numerous pigment granules in various developmental forms (fig. 1). Twenty and sixty-five hundredths percent ( f4.46) of these granules were premelanosomes and 79.28% ( t 4.58) were melanosomes (table 1, fig. 6). At 24 hours after the cells were transferred into in vitro culture, 64.59% ( 2 3 . 9 4 ) of the granules were dense bodies, 2.47% ( t 1.10) were melanosome complexes, 32.14% ( t 2.99) were melanosomes, and less than 1% (0.62% t 0 . 3 0 ) were premelanosomes (figs. 2a,b, 6). In the intact retinal pigment cells of 7-day embryos, more than 86% (86.69% k 4.54) of the pigment granules were melanosomes and 12.07% ( & 4.80) were premelanosomes, showing that melanogenesis had gradually progressed (figs. 3, 6). Less than 2 % (1.23% k0.27) consisted of the dense body-like structures. Actinomycan D (1) Inhibition of transformation of pigment granules Retinal pigment cells from 6-day embryos were cultured for 24 hours in a
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Y. K. TAKEUCHI, I . K . TAKEUCHI A N D T. KAJISHIMA
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INHIBITION OF MELANOSOME TRANSFORMATION TABLE 1
Number of pigment granules of each type in retinal pigment cells of 6-day and ?-day chick embryos, and of untreated and actinomycin D-treated 24-hour cultures Number of each type of pigment granules Sample
Number of cells
Total
M
PM
DB
128 147 150 155
3672 3597 3857 3504
2994 3132 1218 2685
678 413 30 390
0 52 2535 395
MC
~~
6-day embryo 7-day embryo 24-hr, cultured 24-hr, cultured with AD
1
0 0 74 34
1 Number of the structures closely resembling dense bodies observed in the cultured cells. AD, actinomycin D; DB, dense body; M, melanosome; MC, melanosome complex; PM, premelanosome.
TABLE 2
Number of pigment granules of each type in retinal pigment cells of 24-hour cultures treated with nctinomycin D at various times during culture ~
Number of each type of pigment granules AD treatment period (hr)
Number of cells
Total
M
PM
DB
MC
0-4
144 150 150 143
3568 3557 371 1 3502
3100 3080 3020 2747
269 152 202 206
180 309 455 487
19 16 34 62
4-8 8-12 12-24
Abbreviations are the same as in table 1.
medium containing 0.2 pglml of actinomycin D. In these cells about 12% (1 1.69% 2.06) of the granules were dense bodies, while more than 75% (76.67% f2.15) were melanosomes, and 11.69% ( f 2.06) were premelanosomes (table 1 , figs. 4, 6). Thus the number of dense bodies decreased markedly from the untreated 24-hour cultured cells, but the ratio of melanosomes to premelanosomes was similar to that in the intact 7-day embryo cells. From these results, it is evident that actinomycin D prevented the transformation of pigment granules into dense bodies.
*
Fig. 3 Intact retinal pigment cell of a 7-day embryo. M a n y melanosomes (M) are observed in the cytoplasm. C, centriole; Mt, mitochondria; PM, prernelanosome. X 26,500. Fig. 4 Twenty-four-hour cultured cell treated with actinomycin D. Numerous melanosomes (M) are observed, but no dense bodies are observed in the cytoplasm. L, lipid droplet; Mt, mitochondria; N, nucleus. X 16,500. Fig. 5 Phase contrast micrographs of in vitro cultured retinal pigment cells, treated with actinornycin D for the first four hours then transferred to the normal culture medium. 5a: Twenty-fourhour culture. No outgrowth of cells is observed from the explant. X 300. 5b: Seven-day cultured cells showing active outgrowth. The peripheral cells appear to be lightly pigmented, whereas the eentral cells are heavily pigmented. x 1,000.
( 2 ) Duration of inhibitory effects Cells from 6-day embryos were treated with actinomycin D at varying times and durations during the first 24-hour culture period. Table 2 and figure 7 show the results of this treatment. It can be seen that the later the period of actinomycin D treatment the greater the proportion of the dense bodies, and of the melanosome complexes as well (fig. 7). Thus, it was shown actinomycin D had an inhibitory effect on the transformation of pigment granules if the cells were treated early.
(3) Reuersibility of inhibitory effects Cells of 6-day embryos were exposed to 0.2 &ml actinomycin D during the first four hours of culture and then rinsed three times with Tyrode’s solution, and subsequently cultured in normal culture medium for eight days. The explants began outgrowth at four to five days, and the cells changed from a round to an elongated shape (figs. 5a,b). When observed under a light microscope, these elongated cells appeared to be lighter in pigmentation (fig. 5b), and with an electron microscope, numerous dense bodies and melanosome were observed in the cytoplasm.
396
Y. K. TAKEUCHI, I. K. TAKEUCHI AND T. KAJISHIMA I
1001
I -
---=
+AD
-...I.
M PM
DB MC
Fig. 6 Percentage of each type of pigment granules present in retinal pigment cells of 6-day and 7-day chick embryos, and of untreated and actinomycin D-treated 24-hour cultures. Values given represent mean 5 S.D. of 128 cells (6-day embryo), 147 cells (7-day embryo), 150 cells (24-hour-cultured), and 155 cells (24-hour-cultured with actinomycin D), respectively. AD, actinomycin D; DB, dense body; M, melanosome; MC, melanosome complex; PM, premelanosome.
Fig. 7 Percentage of each type of cultures treated with actinomycin D mean ? S.D. of 144 cells (--hour (8-12-hour treatment), and 143 cells the same a s in figure 6.
pigment granules in retinal pigment cells of 24-hour at various periods of culture. Values given represent treatment), 150 cells (4-8-hour treatment), 150 cells (12-24-hour treatment), respectively. Abbreviation i s
The treated continuously tinomych D for 50 to 70 hours from the beginning of the culture showed degenerative profiles and never recover4 even after they were to the normal culture medium; the dense body formation was apparently prevented in these cells.
Colcemid When cells from 6-day embryos were treated with 2 X M colcemid for 24
Fig. 8 Effects of colcemid on cultured retinal pigment cells. 8a: Twenty-four-hour cultured cells. Numerous melanosomes (M) are observed in the cytoplasm. Dense bodies are not encountered. Mt, mitochondria; N, nucleus. X 20,000. 8b: A cell in the peripheral region of 8-day culture. The melanosomes and their demadatine oreanelles have almost completely disappeared &om-the cytoplasm. ER, endoplasmic reticulum; Mt, mitochondria; N, nucleus. X 10,000. 8c: A cell of central area of 8-day culture. Numerous dense bodies (DB) and pigment granules can still be observed. M, melanosome. x 9,500.
INHIBITION OF MELANOSOME TRANSFORMATION
397
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Y . K. TAKEUCHI, I. K. TAKEUCHI A N D T. KAJISHIMA
or 40 hours from the beginning of the culture, few dense bodies and melanosome complexes could be observed in the cytoplasm (fig. 8a). However, cells continuously exposed to colcemid for eight days (the medium was changed every 2 days) exhibited transformation of pigment granules in the cytoplasm, though proliferation of cells was never observed during this period. In cells of the peripheral region, transformation took place as early as the first four days, and pigment granules and their degenerative organelles almost completely disappeared between five and eight days of culture (fig. 8b). On the other hand, in the central parts of the explants degeneration of pigment granules was retarded considerably, dense bodies and melanosome complexes being frequently observed in the cytoplasm even in cells cultured for eight days (fig. 8c). DISCUSSION
and Godman, '71; Soeiro and Amos, '66). However, the inhibitory effects on transformation of pigment granules in the present study do not seem to be caused by the cytotoxic effect of actinomycin D, since transformations were prevented and the cells recovered if they were retransferred to normal culture medium after treatment for four hours. There is little doubt that the d e novo synthesis of DNA-dependent RNA takes place during the initiation of transformation of pigment granules, and that RNA synthesis commences soon after the cells are transferred into culture conditions. In this connection, it is interesting that Whittaker ('68) has reported that RNA synthesis can be detected in retinal pigment cells shortly after plating, and that this RNA synthesis is almost completely blocked by actinomycin D. Although he considered that these rapidly-synthesized RNAs might be used for the protein synthesis required for cell proliferation, it is also plausible that a part of these RNAs may carry information to initiate the transformation of pigment granules, perhaps thereby activating the pre-existing lysosomal enzymes in the pigment granules. In cells which were cultured in colcemidcontaining medium the initiation of transformation of the pigment granules was somewhat delayed compared with the control. Colcemid is known as a mitotic inhibitor. In the present experiment, however, inhibition of proliferation may not have any relationship with the txansformation of pigment granules since depigmentation was generally observed in cell. cultured for a long term with colcemid. Recently it was suggested that the release of lysosomal enzymes was reduced considerably by colchicine (Zurier et al., '73). Therefore it seems reasonable to conclude that the delay of transformation of pigment granules may be caused by such an inhibitory effect of colcemid on the lysosomal enzymes. In any case, we considered the cell proliferation itself to exert no primary effect on the transformation of pigment granules, although it could contribute to the depigmentation in some way such as accelerating the dilution of pigment granules.
In the previous studies, we have presented evidence that the first structural change in monolayer cultured chick retinal pigment cells is the transformation of pigment granules into dense body-like structures (simply designated "dense bodies"), and some of these fused with each other to form melanosome complexes within the first 24 hours. Recent progress in pigment cell biology has revealed that melanin granules in various kinds of pigment cells contain certain lysosomal enzymes such as acid phosphatase (Abraham and Hemdy, '70; Kikuchi, '68; Maul and Romsdahl, '70; Novikoff et al., '68; Olson et al., '70; Seiji and Kikuchi, '69; Takeuchi et al., '71; Wolff and Schreiner, '71), aryl sulphatase (Olson et al., '70), p-N-acetylglucosaminidase (Siakotos et al., '73) and others (Siakotos et al., '73), and that these acid hydrolases may be concerned with the degradation of melanin granules (Maul and Romsdahl, '70; Seiji and Kikuchi, '69). As shown in the present study, the transformation of pigment granules in cultured cells was inhibited considerably by treatment with actinomycin D. Actinomycin D is a well known antibiotic reagent and is an inhibitor of DNA-directed RNA synthesis, but it also has some other efACKNOWLEDGMENT fects on the biosynthetic pathways, includThe authors wish to express their aping cytotoxicity (Honig and Rabinovitz, '65; Laszlo et al.,'66; Revel et al., '64; Sawicki preciation to Mrs. Cherrie A. Mahi of The
INHIBITION OF MELANOSOME TRANSFORMATION
Hawaii University for her critical reading of the manuscript. LITERATURE CITED Abraham, R., and R. J. Hemdy 1970 Irreversible lysosomal damage induced by chloroquine in the retinae of pigmented and albino rats. Exp. Mol. Pathol., 12: 185-200. Cahn, R. D., and M. D. Cahn 1966 Heritability of cellular differentiation: clonal growth and expression of differentiation in retinal pigment cells in vitro. Proc. Nat. Acad. Sci., 55: 106-114. Coulter, H.D. 1967 Rapid and improved methods for embedding biological tissues in Epon 812 and Araldite 502. J. Ultrastruct. Res., 20:
346355. Dalton, A. J. 1955 A chromeosmium fixative for electron microscopy. Anat. Rec., 121: 281. Deitch, A. D., and G. C. Godman 1966 The cytoplasm of the cultured cells treated with actinomycin D. Fed. Proc., 25: 357. Fitzpatrick, T. B., W. C. Quevedo, Jr., A. L. Levene, v . J. McGovern, Y. Mishima and A. G. Oettle 1966 Terminology of vertebrate melanin-containing cells. Science, 152: 88-89. Hamburger, V., and H. L. Hamilton 1951 A series of normal stages in the development of the chick embryo. J. Morph., 88:49-92. Honig, G. R., and M. Rabinovitz 1965 Actinomycin D: inhibition of protein synthesis unrelated to effect on template RNA synthesis. Science, 149: 1504-1506. Kikuchi, A. 1968 Acid phosphatase activity in melanosomes of melanocytes. Bull. Tokyo Med. Dent. Univ., 15: 279-294. Laszlo, J., D. S. Miller, K. S. McCarty and P. Hochstein 1966 Actinomycin D: inhibition of respiration and glycolysis. Science, 151: 1007-1010. Maul, G. G.,and M. M. Romsdahl 1970 Ultrastructural comparison of two human malignant melanoma cell lines. Cancer Res., 30: 2782-
2790. Mishima, Y. 1967 Cellular and subcellular activities in the ontogeny of nevocytic and melanocytic melanomas. In: Advances in Biology of Skin. Vol. 3. The Pigmentary System. W. Montagna and F. Hu, eds. Pergamon Press, London, pp. 50S548. Novikoff, A. B., A. Albara and L. Biempica 1968 Ultrastructural and cytochemical observations on B-16 and Harding-Passey mouse melanomas. The origin of premelanosomes and compound melanosomes. J. Histochem. Cytochem., 16: 299-
319. Olson, R. L., J. Nordquist and M. A. Everett 1970
399
The role of epidermal lysosomes in melanin physiology. Brit. J. Derm., 83: 189-199. Revel, M., H. H. Hiatt and J. Revel 1963 Actinomycin D: an effect on rat liver homogenates unrelated to its action on RNA synthesis. Science, 146: 1311-1313. Sawicki, S . C., and G. C. Godman 1971 On the differential cytotoxicity of actinomycin D. J. Cell Biol., 50: 746761. Seiji, M., and A. Kikuchi 1969 Acid phosphatase activitv in melanosomes. J. Invest. Derm.. 52.. 212-216. Siakotos, A. N., V. Pate1 and A . Cantaboni 1973 The isolation and chemical composition of p r e melanosomes and melanosomes: human and mouse melanomas. Biochem. Med., 7: 14-24. Soeiro, R., and H. Amos 1966 mRNA half-life measured by the use of actinomycin D in animal cells - a caution. Biochem. Biophys. Acta, 129: 406409. Takeuchi, Y . K., I. K. Takeuchi and K. Takata 1971 Electron microscopical studies on the depigmentation of in vitro cultured embryonic chick pigment retina (in Japanese). Zool. Mag., 80: 450. 1973 An electron microscopic observation on the depigmentation process of chick retinal pigment cells cultured in vitro. Annot. Zool. Japon., 4 6 : 154-164. Weiss, P. 1949 Differential growth. In: The Chemistry and Physiology of Growth. A. K. Parpart, ed. Princeton University Press, Princeton, pp. 135-186. Whittaker, J. R. 1963 Changes in melanogenesis during dedifferentiation of chick retinal pigment cells in cell culture. Develop. Biol., 8: ~
99-127. 1967 Loss of melanotic phenotype in vitro by differentiated retinal pigment cells: demonstration of mechanisms involved. Develop. Biol., 15: 553-574. 1968 Translational competition as a possible basis of modulation in retinal pigment cell cultures. J. Exp. Zool., 169: 143-160. 1970 The melanotic expression of embryonic pigment cells: regulation in vitro and in situ. In: Control Mechanisms of Cellulax Phenotypes. Symposia of the International Society for Cell Biology. Vol. 9. H. A. Padykula, ed. Academic Press, New York, pp. 89-108. Wolff, K., and E. Schreiner 1971 Melanosomal acid phosphatase. Arch. Derm. Forsch., 241 :
255-272. Zurier, R. B., S. Hoffstein and G. Weissmann 1973 Mechanisms of lysosomal enzyme release from human leukocytes. I. Effect of cyclic nucleotides and colchicine. J. Cell Biol., 58: 27-41.
ABSTRACT
When the retinal pigment epithelial cells of the chick embryo are transferred to monolayer cultures, they lose their phenotypic trait melanin granules - after a few days. Within the first 24 hours almost all of the melanosomes and premelanosomes are transformed into the degradative structures of the dense bodies or the melanosome complexes. Then, within a few days, these structures disappear completely from the cytoplasm. Actinomycin D, added to the culture medium during the first four hours, almost completely prevents the transformation of melanosomes and premelanosomes. The inhibition of cell proliferation, caused by the addition of colcemid, does not prevent the transformation, though the time of initiation of transformation is delayed considerably. The mechanisms of the transformation of pigment granules are discussed
Retinal pigment cells of the chick embryo lose their phenotypic trait - melanin granules - under conditions of monolayer cell culture (Whittaker, ’63, ’67; Cahn and Cahn, ’66). This fact has been widely known as one of the phenomena of “dedifferentiation,” or, according to Weiss (‘49), “modulation of cell differentiation.” Whittaker (’63, ’67) has investigated the chemical characteristics of the melanin synthesizing systems of retinal pigment cells during depigmentation and found that declines in melanin concentration, tyrosinase activity, and rate of tyrosinase-dependent incorporation of tyrosine are initiated during the f m t few hours of culture. From these results, he has proposed the hypothesis that the cessation of tyrosinase synthesis in the pigment cells and the dilution of pigment granules by cell proliferation are the principal factors contributing to the loss of phenotype. To clarify the mechanisms of regulation of enzyme synthesis, Whittaker ( 68) mvestigated DNA, RNA, and protein synthesis in cultured retinal pigment cells by the method of determining the incorporation rate of labeled precursors into the cells; he found that a new DNA synthesis is not correlated with a loss of tyrosinase activity, whereas a close correlation does exist between a loss of tyrosinase synthesis J. EXP.ZOOL., 192: 391400.
and a rapid increase in the rate of both RNA and protein synthesis. He calculated the methylation ratio (methyl labevuridine label) of ribonuclease-digested RNA and concluded that the rapidly-synthesized RNAs in early cultures involve messenger RNA in addition to ribosomal RNA and transfer RNA. Believing that this messenger RNA might be used for the protein synthesis required for cell proliferation, he proposed the hypothesis of socalled “translational competition” between de novo synthesized messenger RNA and the preexisting tyrosinase messenger RNA. In recent electron microscopy studies we have demonstrated that the primary morphological change of this depigmentation is the transformation of pigment granules (Takeuchi et al., ’73). Within 24 hours after transferring the cells into culture, numerous electron-dense bodies (simply designated “dense bodies”) and melanosome complexes (probably formed by fusion of dense bodies with slowly degradating pigment granules) appeared in the cytoplasm. Conversion of pigment granules into dense bodies and melanosome complexes was followed by ultrastructural observation and by electron microscopic autora1 Present address: Department of Embryology, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan.
391
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INHIBITION OF MELANOSOME TRANSFORMATION
diography of 3H-Dopa incorporation. These results are in perfect accord with the depigmentation process of in vitro cultured mammalian melanoma cells (Mishima, '67; Novikoff et al., '68). However, what is involved in initiating the transformation of pigment granules in these cells remains unknown. In the present study, we used electron microscopy to examine the effects of actinomycin D and colcemid on the transformation of pigment granules. We hoped thereby to demonstrate the principal factor(s) required for initiating the depigmentation of retinal pigment cells in vitro. The nomenclature suggested by Fitzpatrick et al. ('66) for the terminology of pigment granules is used with slight modifications in this paper. MATERIALS AND METHODS
Cell culture Samples of retinal pigment epighelium from 6-day White Leghorn chick embryos (Hamburger-Hamilton's stage 28 [Hamburger and Hamilton, '511) were cut into pieces smaller than 1 X 1 mm, inoculated onto plasma clots (chick embryonic extract; chicken plasma [Difco], l : l ) , and grown as monolayer sheets. The culture medium consisted of equal parts of Tyrode's solution, horse serum, and 9-day chick embryonic extract. Penicilin G and dihydrostreptomycin were added to this medium. The methods of preparation and culture used in the present study were the same as those previously described (Takeuchi et al., '73). Treatments Actinomycin D (Merck) was added to the culture medium at a final concentration of 0.2 pglml. Colcemid (K & K Laboratories, Inc.) was used at a concentration of 2 X M. Fig. 1 Intact retinal pigment cell of a 6-day chick embryo. Numerous pigment granules in various developmental stages are observed. M, melanosome; Mt, mitochondria; N, nucleus; PM, premelanosome. X 22,800. Fig. 2 Twenty-four-hour cultured cells. 2a: Numerous dense bodies (DB) are observed in the cytoplasm. Nucleolus (No) becomes prominent. M, melanosome; N , nucleus. X 15,500. 2b: Melanosome complex (MC) contains many pigment granules. x 16,000.
393
Electron microscopy The cells were collected with a rubber policeman and fixed with Dalton's chromeosmium fixative (Dalton, '55) for two hours at 4°C. Dehydration was performed with an ethanol series and propylene oxide, then the specimens were embedded in Epon 812 according to Coulter ('67). Thin sections were cut on a Porter-Blum MT-2 ultramicrotome, double-stained with uranyl acetate and lead nitrate, and examined with a Hitachi HU 11-DS electron microscope at 75 kV. To obtain the definitive evidence, pigment granules were classified into four types - as premelanosome, melanosome (including the melanin granule described by Fitzpatrick et al., '66), dense body, and melanosome complex, and the pigment granules present in each cell were counted according to their respective types. The counts were performed on approximately 150 cells at each experimental stage. RESULTS
Control Intact retinal pigment cells from 6-day chick embryos contained numerous pigment granules in various developmental forms (fig. 1). Twenty and sixty-five hundredths percent ( f4.46) of these granules were premelanosomes and 79.28% ( t 4.58) were melanosomes (table 1, fig. 6). At 24 hours after the cells were transferred into in vitro culture, 64.59% ( 2 3 . 9 4 ) of the granules were dense bodies, 2.47% ( t 1.10) were melanosome complexes, 32.14% ( t 2.99) were melanosomes, and less than 1% (0.62% t 0 . 3 0 ) were premelanosomes (figs. 2a,b, 6). In the intact retinal pigment cells of 7-day embryos, more than 86% (86.69% k 4.54) of the pigment granules were melanosomes and 12.07% ( & 4.80) were premelanosomes, showing that melanogenesis had gradually progressed (figs. 3, 6). Less than 2 % (1.23% k0.27) consisted of the dense body-like structures. Actinomycan D (1) Inhibition of transformation of pigment granules Retinal pigment cells from 6-day embryos were cultured for 24 hours in a
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INHIBITION OF MELANOSOME TRANSFORMATION TABLE 1
Number of pigment granules of each type in retinal pigment cells of 6-day and ?-day chick embryos, and of untreated and actinomycin D-treated 24-hour cultures Number of each type of pigment granules Sample
Number of cells
Total
M
PM
DB
128 147 150 155
3672 3597 3857 3504
2994 3132 1218 2685
678 413 30 390
0 52 2535 395
MC
~~
6-day embryo 7-day embryo 24-hr, cultured 24-hr, cultured with AD
1
0 0 74 34
1 Number of the structures closely resembling dense bodies observed in the cultured cells. AD, actinomycin D; DB, dense body; M, melanosome; MC, melanosome complex; PM, premelanosome.
TABLE 2
Number of pigment granules of each type in retinal pigment cells of 24-hour cultures treated with nctinomycin D at various times during culture ~
Number of each type of pigment granules AD treatment period (hr)
Number of cells
Total
M
PM
DB
MC
0-4
144 150 150 143
3568 3557 371 1 3502
3100 3080 3020 2747
269 152 202 206
180 309 455 487
19 16 34 62
4-8 8-12 12-24
Abbreviations are the same as in table 1.
medium containing 0.2 pglml of actinomycin D. In these cells about 12% (1 1.69% 2.06) of the granules were dense bodies, while more than 75% (76.67% f2.15) were melanosomes, and 11.69% ( f 2.06) were premelanosomes (table 1 , figs. 4, 6). Thus the number of dense bodies decreased markedly from the untreated 24-hour cultured cells, but the ratio of melanosomes to premelanosomes was similar to that in the intact 7-day embryo cells. From these results, it is evident that actinomycin D prevented the transformation of pigment granules into dense bodies.
*
Fig. 3 Intact retinal pigment cell of a 7-day embryo. M a n y melanosomes (M) are observed in the cytoplasm. C, centriole; Mt, mitochondria; PM, prernelanosome. X 26,500. Fig. 4 Twenty-four-hour cultured cell treated with actinomycin D. Numerous melanosomes (M) are observed, but no dense bodies are observed in the cytoplasm. L, lipid droplet; Mt, mitochondria; N, nucleus. X 16,500. Fig. 5 Phase contrast micrographs of in vitro cultured retinal pigment cells, treated with actinornycin D for the first four hours then transferred to the normal culture medium. 5a: Twenty-fourhour culture. No outgrowth of cells is observed from the explant. X 300. 5b: Seven-day cultured cells showing active outgrowth. The peripheral cells appear to be lightly pigmented, whereas the eentral cells are heavily pigmented. x 1,000.
( 2 ) Duration of inhibitory effects Cells from 6-day embryos were treated with actinomycin D at varying times and durations during the first 24-hour culture period. Table 2 and figure 7 show the results of this treatment. It can be seen that the later the period of actinomycin D treatment the greater the proportion of the dense bodies, and of the melanosome complexes as well (fig. 7). Thus, it was shown actinomycin D had an inhibitory effect on the transformation of pigment granules if the cells were treated early.
(3) Reuersibility of inhibitory effects Cells of 6-day embryos were exposed to 0.2 &ml actinomycin D during the first four hours of culture and then rinsed three times with Tyrode’s solution, and subsequently cultured in normal culture medium for eight days. The explants began outgrowth at four to five days, and the cells changed from a round to an elongated shape (figs. 5a,b). When observed under a light microscope, these elongated cells appeared to be lighter in pigmentation (fig. 5b), and with an electron microscope, numerous dense bodies and melanosome were observed in the cytoplasm.
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Y. K. TAKEUCHI, I. K. TAKEUCHI AND T. KAJISHIMA I
1001
I -
---=
+AD
-...I.
M PM
DB MC
Fig. 6 Percentage of each type of pigment granules present in retinal pigment cells of 6-day and 7-day chick embryos, and of untreated and actinomycin D-treated 24-hour cultures. Values given represent mean 5 S.D. of 128 cells (6-day embryo), 147 cells (7-day embryo), 150 cells (24-hour-cultured), and 155 cells (24-hour-cultured with actinomycin D), respectively. AD, actinomycin D; DB, dense body; M, melanosome; MC, melanosome complex; PM, premelanosome.
Fig. 7 Percentage of each type of cultures treated with actinomycin D mean ? S.D. of 144 cells (--hour (8-12-hour treatment), and 143 cells the same a s in figure 6.
pigment granules in retinal pigment cells of 24-hour at various periods of culture. Values given represent treatment), 150 cells (4-8-hour treatment), 150 cells (12-24-hour treatment), respectively. Abbreviation i s
The treated continuously tinomych D for 50 to 70 hours from the beginning of the culture showed degenerative profiles and never recover4 even after they were to the normal culture medium; the dense body formation was apparently prevented in these cells.
Colcemid When cells from 6-day embryos were treated with 2 X M colcemid for 24
Fig. 8 Effects of colcemid on cultured retinal pigment cells. 8a: Twenty-four-hour cultured cells. Numerous melanosomes (M) are observed in the cytoplasm. Dense bodies are not encountered. Mt, mitochondria; N, nucleus. X 20,000. 8b: A cell in the peripheral region of 8-day culture. The melanosomes and their demadatine oreanelles have almost completely disappeared &om-the cytoplasm. ER, endoplasmic reticulum; Mt, mitochondria; N, nucleus. X 10,000. 8c: A cell of central area of 8-day culture. Numerous dense bodies (DB) and pigment granules can still be observed. M, melanosome. x 9,500.
INHIBITION OF MELANOSOME TRANSFORMATION
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Y . K. TAKEUCHI, I. K. TAKEUCHI A N D T. KAJISHIMA
or 40 hours from the beginning of the culture, few dense bodies and melanosome complexes could be observed in the cytoplasm (fig. 8a). However, cells continuously exposed to colcemid for eight days (the medium was changed every 2 days) exhibited transformation of pigment granules in the cytoplasm, though proliferation of cells was never observed during this period. In cells of the peripheral region, transformation took place as early as the first four days, and pigment granules and their degenerative organelles almost completely disappeared between five and eight days of culture (fig. 8b). On the other hand, in the central parts of the explants degeneration of pigment granules was retarded considerably, dense bodies and melanosome complexes being frequently observed in the cytoplasm even in cells cultured for eight days (fig. 8c). DISCUSSION
and Godman, '71; Soeiro and Amos, '66). However, the inhibitory effects on transformation of pigment granules in the present study do not seem to be caused by the cytotoxic effect of actinomycin D, since transformations were prevented and the cells recovered if they were retransferred to normal culture medium after treatment for four hours. There is little doubt that the d e novo synthesis of DNA-dependent RNA takes place during the initiation of transformation of pigment granules, and that RNA synthesis commences soon after the cells are transferred into culture conditions. In this connection, it is interesting that Whittaker ('68) has reported that RNA synthesis can be detected in retinal pigment cells shortly after plating, and that this RNA synthesis is almost completely blocked by actinomycin D. Although he considered that these rapidly-synthesized RNAs might be used for the protein synthesis required for cell proliferation, it is also plausible that a part of these RNAs may carry information to initiate the transformation of pigment granules, perhaps thereby activating the pre-existing lysosomal enzymes in the pigment granules. In cells which were cultured in colcemidcontaining medium the initiation of transformation of the pigment granules was somewhat delayed compared with the control. Colcemid is known as a mitotic inhibitor. In the present experiment, however, inhibition of proliferation may not have any relationship with the txansformation of pigment granules since depigmentation was generally observed in cell. cultured for a long term with colcemid. Recently it was suggested that the release of lysosomal enzymes was reduced considerably by colchicine (Zurier et al., '73). Therefore it seems reasonable to conclude that the delay of transformation of pigment granules may be caused by such an inhibitory effect of colcemid on the lysosomal enzymes. In any case, we considered the cell proliferation itself to exert no primary effect on the transformation of pigment granules, although it could contribute to the depigmentation in some way such as accelerating the dilution of pigment granules.
In the previous studies, we have presented evidence that the first structural change in monolayer cultured chick retinal pigment cells is the transformation of pigment granules into dense body-like structures (simply designated "dense bodies"), and some of these fused with each other to form melanosome complexes within the first 24 hours. Recent progress in pigment cell biology has revealed that melanin granules in various kinds of pigment cells contain certain lysosomal enzymes such as acid phosphatase (Abraham and Hemdy, '70; Kikuchi, '68; Maul and Romsdahl, '70; Novikoff et al., '68; Olson et al., '70; Seiji and Kikuchi, '69; Takeuchi et al., '71; Wolff and Schreiner, '71), aryl sulphatase (Olson et al., '70), p-N-acetylglucosaminidase (Siakotos et al., '73) and others (Siakotos et al., '73), and that these acid hydrolases may be concerned with the degradation of melanin granules (Maul and Romsdahl, '70; Seiji and Kikuchi, '69). As shown in the present study, the transformation of pigment granules in cultured cells was inhibited considerably by treatment with actinomycin D. Actinomycin D is a well known antibiotic reagent and is an inhibitor of DNA-directed RNA synthesis, but it also has some other efACKNOWLEDGMENT fects on the biosynthetic pathways, includThe authors wish to express their aping cytotoxicity (Honig and Rabinovitz, '65; Laszlo et al.,'66; Revel et al., '64; Sawicki preciation to Mrs. Cherrie A. Mahi of The
INHIBITION OF MELANOSOME TRANSFORMATION
Hawaii University for her critical reading of the manuscript. LITERATURE CITED Abraham, R., and R. J. Hemdy 1970 Irreversible lysosomal damage induced by chloroquine in the retinae of pigmented and albino rats. Exp. Mol. Pathol., 12: 185-200. Cahn, R. D., and M. D. Cahn 1966 Heritability of cellular differentiation: clonal growth and expression of differentiation in retinal pigment cells in vitro. Proc. Nat. Acad. Sci., 55: 106-114. Coulter, H.D. 1967 Rapid and improved methods for embedding biological tissues in Epon 812 and Araldite 502. J. Ultrastruct. Res., 20:
346355. Dalton, A. J. 1955 A chromeosmium fixative for electron microscopy. Anat. Rec., 121: 281. Deitch, A. D., and G. C. Godman 1966 The cytoplasm of the cultured cells treated with actinomycin D. Fed. Proc., 25: 357. Fitzpatrick, T. B., W. C. Quevedo, Jr., A. L. Levene, v . J. McGovern, Y. Mishima and A. G. Oettle 1966 Terminology of vertebrate melanin-containing cells. Science, 152: 88-89. Hamburger, V., and H. L. Hamilton 1951 A series of normal stages in the development of the chick embryo. J. Morph., 88:49-92. Honig, G. R., and M. Rabinovitz 1965 Actinomycin D: inhibition of protein synthesis unrelated to effect on template RNA synthesis. Science, 149: 1504-1506. Kikuchi, A. 1968 Acid phosphatase activity in melanosomes of melanocytes. Bull. Tokyo Med. Dent. Univ., 15: 279-294. Laszlo, J., D. S. Miller, K. S. McCarty and P. Hochstein 1966 Actinomycin D: inhibition of respiration and glycolysis. Science, 151: 1007-1010. Maul, G. G.,and M. M. Romsdahl 1970 Ultrastructural comparison of two human malignant melanoma cell lines. Cancer Res., 30: 2782-
2790. Mishima, Y. 1967 Cellular and subcellular activities in the ontogeny of nevocytic and melanocytic melanomas. In: Advances in Biology of Skin. Vol. 3. The Pigmentary System. W. Montagna and F. Hu, eds. Pergamon Press, London, pp. 50S548. Novikoff, A. B., A. Albara and L. Biempica 1968 Ultrastructural and cytochemical observations on B-16 and Harding-Passey mouse melanomas. The origin of premelanosomes and compound melanosomes. J. Histochem. Cytochem., 16: 299-
319. Olson, R. L., J. Nordquist and M. A. Everett 1970
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The role of epidermal lysosomes in melanin physiology. Brit. J. Derm., 83: 189-199. Revel, M., H. H. Hiatt and J. Revel 1963 Actinomycin D: an effect on rat liver homogenates unrelated to its action on RNA synthesis. Science, 146: 1311-1313. Sawicki, S . C., and G. C. Godman 1971 On the differential cytotoxicity of actinomycin D. J. Cell Biol., 50: 746761. Seiji, M., and A. Kikuchi 1969 Acid phosphatase activitv in melanosomes. J. Invest. Derm.. 52.. 212-216. Siakotos, A. N., V. Pate1 and A . Cantaboni 1973 The isolation and chemical composition of p r e melanosomes and melanosomes: human and mouse melanomas. Biochem. Med., 7: 14-24. Soeiro, R., and H. Amos 1966 mRNA half-life measured by the use of actinomycin D in animal cells - a caution. Biochem. Biophys. Acta, 129: 406409. Takeuchi, Y . K., I. K. Takeuchi and K. Takata 1971 Electron microscopical studies on the depigmentation of in vitro cultured embryonic chick pigment retina (in Japanese). Zool. Mag., 80: 450. 1973 An electron microscopic observation on the depigmentation process of chick retinal pigment cells cultured in vitro. Annot. Zool. Japon., 4 6 : 154-164. Weiss, P. 1949 Differential growth. In: The Chemistry and Physiology of Growth. A. K. Parpart, ed. Princeton University Press, Princeton, pp. 135-186. Whittaker, J. R. 1963 Changes in melanogenesis during dedifferentiation of chick retinal pigment cells in cell culture. Develop. Biol., 8: ~
99-127. 1967 Loss of melanotic phenotype in vitro by differentiated retinal pigment cells: demonstration of mechanisms involved. Develop. Biol., 15: 553-574. 1968 Translational competition as a possible basis of modulation in retinal pigment cell cultures. J. Exp. Zool., 169: 143-160. 1970 The melanotic expression of embryonic pigment cells: regulation in vitro and in situ. In: Control Mechanisms of Cellulax Phenotypes. Symposia of the International Society for Cell Biology. Vol. 9. H. A. Padykula, ed. Academic Press, New York, pp. 89-108. Wolff, K., and E. Schreiner 1971 Melanosomal acid phosphatase. Arch. Derm. Forsch., 241 :
255-272. Zurier, R. B., S. Hoffstein and G. Weissmann 1973 Mechanisms of lysosomal enzyme release from human leukocytes. I. Effect of cyclic nucleotides and colchicine. J. Cell Biol., 58: 27-41.
