Ultrastructural Study of the Persistence of Colchicine-Induced Cytological Changes in Harding-Passey Melanoma1

[CANCER RESEARCH 38. 168-176, January 1978]

Ultrastructural Study of the Persistence of Colchicine-induced Cytological Changes in Harding-Passey Melanoma1

Kenneth R. Loader and Edward J. H. Nathaniel2

Department ol Anatomy, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

ABSTRACT and characterizing the differentiated state. Since they are not always concordant, it becomes important to recognize Electron microscopic observations were undertaken on the significance of each, and to specify criteria when one Harding-Passey melanoma in control tumors, colchicine- is characterizing differentiation. Cells may be said to be treated tumors, and tumors that were passed through differentiated by morphological, behavioral, chemical or five transplantations in mice subsequent to colchicine developmental criteria." treatment. Evidence of submicroscopic differentiation is observed The untreated Harding-Passey melanoma tumor was in both the nucleus and the cytoplasm. These changes are composed of pleomorphic cells the cytoplasm of which more conspicuous in the cytoplasm (13). Morphological contained tubulovesicular mitochondria and numerous changes in the nucleus during the course of development free and membrane-attached ribosomes. Type A virus have been described by several investigators (3, 22). How particles were observed in both mitotic and interphase ever, the site of functional realization of the cell, together cells. Colchicine-treated tumors exhibited several typical with the respective structural arrangement, resides within colchicine mitotic cells arrested in metaphase. A most the cytoplasm, the primary area of submicroscopic differ significant feature of the colchicine administration was entiation. During differentiation, changes in cellular organ- the appearance of microfilaments in both interphase and elles take place. mitotic cells, a feature not observed in cells of the un The appearance of microfilaments in the tumor cells of treated tumor. the Harding-Passey melanoma following colchicine treat Electron microscopy of colchicine-treated tumors car ment was first reported by Nathaniel et al. (37). The present ried through five transplantations at biweekly intervals study was designed to establish whether the microfilaments revealed the presence of microfilaments in tissues fixed are transitory or persist through successive generations of at each of the transplantations. tumor transfer without further treatment. It therefore appears that the cytological effects of col This report presents the ultrastructural features of the chicine resulting in the appearance of filaments in mela- Harding-Passey melanoma following colchicine administra nocytes of the Harding-Passey melanoma are more en tion and compares these with those of the untreated control during than they were initially presumed to be. tumor. Subsequently, the cytology of the treated tumor is described as the tumor is passed through 5 transplantations INTRODUCTION in mice, with special attention paid to the presence of filaments. Whereas the beneficial effect of X-rays and radioactive isotopes in the treatment of human cancers has been definitely established, there remain certain types of tumors MATERIALS AND METHODS that are radioresistant, and melanomas belong to this Female BALB/cJ mice bearing the Harding-Passey mela group. Consequently, the effects of a number of chemical noma received i.p. injections of colchicine in doses of 0.5 compounds and antibiotics on different animal melanomas mg/kg daily for 7 days starting 1 day after tumor transplan are being investigated by several researchers (11, 45, 56). tation. A further dose of 1.0 mg/kg was given daily for 7 Suguria (52), following an extensive study on the inhibitory more days. All animals were sacrificed 24 hr after the last effects of chemical compounds and antibiotics on the injection, tumors were exposed by blunt dissection, and Harding-Passey melanoma, observed that alkylating agents small pieces of tumor were then transplanted to new host and antiobiotics were equally effective. These chemical animals. The remaining treated tumor tissue was fixed in agents exert their beneficial effect not only by their tumori- buffered paraformaldehyde and glutaraldehyde according cidal action but also by promoting cellular maturation and to the method of Karnovsky and further fixed in 2% osmium differentiation. Grobstein (19) defined cytodifferentiation thusly: "Since tetroxide in 0.2 M cacodylate with 0.4% sucrose for 1 hr. Following dehydration, tissues were embedded in Araldite. differentiation affects the cell in many ways and at many This fixed tumor is referred to as "treated tumor." After a levels, there are a number of sets of criteria for identifying 2-week growing period in the host animal, the tumor was removed and the above procedure of transplantation and fixation for electron microscopy was again performed. This ' Supported in part by the National Cancer Institute of Canada and the tumor tissue is referred to as "treated tumor generation 1." Medical Research Council of Canada. 2 To whom requests for reprints should be addressed, at Department of The procedure of transferring the tumor was repeated 4 Anatomy, School of Medicine, 730 William Ave , Winnipeg. Manitoba. Can more times at 2-week intervals, and the resultant tissues ada R3E OW3. are referred to as "treated tumor generations 2, 3, 4, and 5." Received February 14, 1977; accepted October 11, 1977.

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Sections 0.5 /urn thick were cut from the Araldite blocks encountered in interphase cells. Such collections of micro- and stained with toluidine blue. Nonnecrotic areas of tumor filaments may be located anywhere within the cytoplasm of were selected by light microscopy; thin sections then cut the tumor cell (Fig. 4). However, they tend to be perinuclear and stained with uranyl acetate and lead citrate. Sections (Fig. 8). In fact, we often located filament bundles by were mounted on uncoated copper grids and visualized searching the perinuclear region. with the Philips EM 300 electron microscope. A second pattern of microfilament distribution varied from a less defined bundle of filaments (Fig. 3) to a rather RESULTS diffuse, random permeation of the cytoplasm by these filaments (Figs. 5 to 7). In Fig. 8 one observes both patterns Morphology of Untreated Tumors. Electron microscopic of filament distribution; whereas a broad bundle of filaments observations confirmed the optical microscopic findings borders 1 side of the nucleus, the rest of the cytoplasm on that the tumor was highly cellular and that the cells were the other side of the nucleus displays a less defined and extremely pleomorphic (Fig. 1). Melanin granules were more diffuse pattern of distribution of microfilaments. observed in all the cells of the tumor and formed the basis Both the localized and diffuse patterns of microfilament for identification of these cells as melanocytes. The amount distribution were obeserved in both interphase and mitotic of free and attached ribosomes varied in the melanocytes, cells. However, the localized pattern was more commonly accounting for light to heavy basophilia in toluidine blue- observed in interphase cells, whereas the diffuse pattern stained 0.5-/am-thick sections. The cytoplasm revealed tub- was prevalent in mitotic cells. ulovesicular mitochondria and multiple Golgi complexes. Similar to the colchicine-treated tumor, the cells of Virus-like particles were observed in relation to the granular treated tumor generations 1 through 5 displayed consider endoplasmic reticulum of many tumor cells. The tumor able pigment within their cytoplasm. The pigment granules nuclei were prominent and exhibited considerable accu tend to locate principally along the peripheral aspect of the mulation of chromatin along their margin. Mitotic cells in cell (Figs. 4 and 6). all phases of mitosis were observed in the tumor. As indicated earlier the microfilament aggregates were Morphology of Colchicine-treated Tumor. The most strik invariably limited by ribosomal particles, suggesting their ing feature of colchicine treatment was the presence of possible role in filament production. Cytoplasmic organ numerous colchicine mitotic cells. The colchicine mitotic elles and viral inclusions in cells of treated tumor and cell was enlarged and comprised of a central portion of treated tumor generations 1 through 4 resembled the un chromosomes in a dense cytoplasmic matrix. The periph treated tumor. eral portion of the cell generally exhibited dilated cisternae of endoplasmic reticulum. DISCUSSION A significant feature observed in tumor cells treated with colchicine was the presence of microfilaments in both the The effects of chemical analog in experimental animal mitotic and interphase cells. The microfilaments were found tumors are being investigated extensively with the hope of in bundles, with ribosomal rosettes often located outside obtaining information that may be applicable to the treat the bundles rather than among the filaments (Fig. 2). In the ment of human neoplasms. This is especially true of tumors interphase cells the microfilaments were more often peri- that are not sensitive to radiation and are not amenable to nuclear. In addition an increase in melanin granules with a surgery. Many of the chemotherapeutic agents are thought tendency for marginal localization was evident in interphase to exert a beneficial effect by altering the cellular cycle of cells. growth, division, and differentiation. Morphology of Treated Tumor Generations 1 through 5. The effects of colchicine on the microtubules of the The cytological features observed in the cells of the colchi- mitotic cell have been recognized since the work of Eigsti cine-treated tumor were also observed in the melanoma and Dustin (14). The morphology of spindle tubules follow cells throughout the successive tumor generations, 1 ing colchicine therapy has been described by Harris and through 5. Mazia (21) in sea urchin eggs, Brinkley ef al. (4) in the The most striking feature was the continued presence of Chinese hamster cell, and Dougherty and Lee (12) in divid filaments in tumor cells from generation 1 through 5 (Figs. ing rat hepatic cells. 3 to 8). The microfilaments were observed in both mitotic Electron microscopic studies on Cloudman S-91 mouse and interphase cells in all tumor generations studied. In melanoma (8) and Harding-Passey melanoma (37, 48) have general the distribution of the microfilaments within the not revealed the presence of microfilaments in tumor cells. cytoplasm and the size of filament aggregates appeared to Nathaniel ef al. (37) reported the appearance of filaments be similar in tumor cells of all generations. in Harding-Passey melanoma tumor cells after colchicine There were 2 patterns of distribution of microfilaments. treatment. In several cells well-defined, conspicuous bundles of fila The administration of colchicine to mice bearing the ments were encountered (Figs. 4 and 8). Conspicuous Harding-Passey melanoma tumor in this study resulted in collections of polysomes and granular endoplasmic reticu several cytological changes in the tumor cells (29). The lum often bordered such aggregates of filaments (Figs. 4 most striking feature was the appearance in the tumor cells and 8). Cell organelles such as mitochondria and cell of microfilaments, which were not observed in untreated products such as melanin granules were found generally cells. The second important feature was the continued bordering the filament aggregates and rarely among them. presence of the filaments in these tumor cells even after 5 These circumscribed bundles of filaments were more often transplantations over a 10-week period. The third feature

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. K. R. Loader and E. J. H. Nathaniel was the tendency for melanin granules to marginate or be Investigations of the effects of colchicine on the melano- located along the peripheral aspect of the cell. The signifi phores of Fundulus heteroclitus showed that there is a cance of these cytological observations will be discussed. decrease in the microtubules of these cells associated with Colchicine breaks down microtubules in dividing cells an increase in microfilaments and dispersion of melano- (43, 46) and in interphase cells (30, 32, 55). This action is somes (59). Studies by Jande (26) and McGuire (36) also brought about by colchicine binding to sites of interaction indicated that microtubules were primarily concerned with between protein subunits tubulin, the 65 diametric subunits aggregation of melanin granules, whereas their disassem of microtubules, thereby preventing their assembly into bly into microfilaments aided dispersion. microtubules (49, 57). The presence of microfilaments in human epidermal Numerous investigations have shown that both colchicine melanocytes has been reported (6, 39). Jimbow ef al. (27) and vinblastine induce the appearance in cells of filaments conducted a detailed study directed to characterization of measuring 10 nm in diameter (2). Vinblastine, which is also ultrastructural events in melanocytes before and after ex a mitotic inhibitor, inactivates tubules by precipitating tu posure to sun in caucasoid, mongoloid, and negroid sub- bulin as large crystals (2, 33). Nathaniel ef al. (37) observed jets. Both microtubules and microfilaments were observed the appearance of filaments in tumor cells of Harding- in both exposed and unexposed skin. In the unexposed Passey melanoma after colchicine administration. In this skin the microfilaments formed a distinct bundle, which communication the persistence of colchicine-induced fila was invariably perinuclear. After exposure to sunlight and ments in tumor cells of Harding-Passey melanoma over a uv of long wavelength, the microfilaments were located 10-week period is presented. Schubert ef al. (47) reported primarily in the dendritic processes, closely associated with the presence of microfilaments following colchicine and melanosomes. In addition the microtubules were more vinblastine treatment during in vitro differentiation of a frequently located along the periphery of the cytoplasm or mouse neuroblastoma. in the dendritic processes. The functional roles of microfilaments and microtubules The role of microfilaments and microtubules in the dis have been investigated with respect to cell shape altera persion and aggregation of melanin granules has been tions, axonal growth, muscle contraction, and many other very convincingly demonstrated by investigators mentioned cell processes. Wessells ef al. (58) and Spooner and Wessels previously. However, the actual mechanism by which the (51) ascribed to the microfilaments and microtubules a pigment granules are translocated is still obscure. Un contractile function responsible for a broad spectrum of treated tumor cells of the Harding-Passey melanoma con cellular movements and developmental processes. tained few microtubules and no microfilaments. Following The breakdown or dissolution of microtubules by colchi colchicine administration both mitotic and interphase cells cine is connected with cessation of intracellular particle displayed microfilaments. Well-defined bundles of filaments movement and has been observed in helizoan axopods were more commonly encountered in interphase cells. The (54), HeLa cells (15), neurons (28), and other tissues. treated tumor cells also showed a tendency for the melanin Investigations by Yamada ef al. (61) and Daniels (9) on the granules to be located along the peripheral aspect of the effects of colchicine on neurons in vitro suggest that melanoma cells. Light microscopic studies by Freidman microtubules are essential for growth and maintenance of and Drutz (16) reported a similar localization of melanin neurites. granules in tumor cells of the Harding-Passey melanoma The alteration of skin color in fish, amphibians, and after irradiation and nitrogen mustard treatment. In the reptiles as a protective adaptation is well known. This light of the observations of Malawista (31), McGuire and change in skin color is associated with the intracellular Moellmann (34), and Jimbow ef al. (27), one may speculate movement of melanin granules in the dermal and epidermal that the colchicine-induced microfilaments observed in this melanocytes. When pigment granules are dispersed the study probably are responsible for the peripheral displace skin is dark, and skin is light when pigment granules are ment of the melanin granules. aggregated (23). Fujii and Novales (17) observed filaments An extensive increase in neurofilaments in nerve cells in the melanocytes of fish and suggested that they play a has been observed following colchicine administration (60). role in the movement of melanin granules. McGuire ef al. This increase in neurofilaments is reported to be associated (35) are of the opinion that microfilaments, which are with inhibition of axoplasmic transport of monoamines (7) abundant in frog epidermal melanocytes, are responsible and protein (18, 28). Hansson and Norstrom (20) observed for the dispersion of the melanin granules. McGuire and an increase in tangles of filaments in the glial cells of the Moellmann (34) showed that dispersion of melanin granules rat hypothalamohypophyseal system after colchicine ad by melanocyte-stimulating hormone was associated with ministration. the appearance of large numbers of filaments within the Microfilaments have been reported in a variety of cells melanocyte dendritic processes. On addition of cytocha- performing diverse functions. The basal cells of the epider lasin B, the filaments disappeared and the granules reag- mis of adult Rana pipiens (42), as well as the basal cells of gregated. Cytochalasin B also reversed the dispersion of human esophagus and trachea (44), contain an abundance melanin granules induced by cyclic 3':5'-adenosine mono- of filaments. Parakkal and Alexander (41) have offered an phosphate and theophylline (38). On the basis of these excellent review of vertebrate epithelia at the ultrastructural studies, Malawista (31), McGuire and Moellmann (34), and level. Jande (26) considered the filaments observed in Novales and Novales (38) suggested that the intracyto- darkened epidermal melanocytes of R. pipiens to be kera plasmic filaments were the mediators of melanin granule tin. McGuire and Moellmann (34) reported that microfila- dispersion. ments that caused dispersion of melanosomes by melano-

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. Chemotherapy of Mouse Melanoma cyte-stimulating hormone could be distinguished from sim Pigmented S-91 Mouse Melanomas by Phenylacetate and the Possible Related Effects on Growth. J. Nati. Cancer Inst., 30: 611-633, 1963 ilar filaments in keratinocytes by using cytochalasin B. 12. Dougherty. W. J., and Lee. M. M. Light and Electron Microscopic Addition of cytochalasin B resulted in disappearance of Studies of Smooth Endoplasmi Reticulum in Dividing Rat Hepatic microfilaments and aggregation of melanin granules, Cells. J. Ultrastruct. Res., 79. 200-220, 1967. 13. Dvorak. M. Submicroscopic Differentiation. Berlin, Heidelberg. New whereas the filaments in keratinocytes were not affected by York: Springer-Verlag. 1971. cytochalasin B. The filaments observed in the tumor cells 14. Eigsti. O. J.. and Dustin, P. Colchicine in Agriculture, Medicine. Biology of the colchicine-treated Harding-Passey melanoma in the and Chemistry. Ames, Iowa: Iowa State College Press, 1955. 15. Freed, J. J.. Bhisey, A. N., and Lebowitz, M. M. The Relation of present study resemble the microfilaments observed in Microtubules and Microfilaments to the Motility of Cultured Cells. J. melanocytes by several investigators cited earlier. Ozzello Cell Biol..39: 46-47A, 1968. (40) described filaments permeating the cytoplasm as per- 16. Friedman, N. B., and Drutz, E. The Effects of Chemotherapy and Irradiation Therapy on the Differentiation of Experimental Tumors. inuclear bundles in mammary carcinoma cells and sug Cancer. 77 1060-1069. 1968. 17. Fujii, R., and Novales, R. R. Cellular Aspects of the Control of Physiolog gested that they might be contractile proteins. ical Color Changes in Fishes. Am. Zool., 9: 453-464, 1969 Glucocorticoids affect cellular differentiation in develop 18 Grafstein, B.. McEwen, B. S.. and Shelanski, M. L. Axonal Transport of ing lung by slowing cell division and thereby promoting Neurotubule Protein. Nature, 277 289-290, 1970. cell maturation and differentiation (5). Smith ef al. (50) 19. Grobstein. C. Differentiation of Vetebrate Cells In: J BrÃ¤chetand A E Mirsky (eds.). The Cell, Vol. 1. New York: Academic Press. Inc.. 1959. reported that cortisone applied to cultures of fetal lung 20. Hansson, A., and Norstrom, A. Glical Reactions Induced by Colchicine- when epithelial cells were in growth phase promoted Treatment of the Hypothalamic-Neurohypophyseal System. Z. Zell- forsch. Mikroscope. Anat. 773: 294-310. 1977. growth. On the other hand, they observed that, when the 21. Harris, P., and Mazia. D. The Finer Structure of the Mitotic Apparatus. steroid was applied to more mature fetal lung, cortisone Symp. Intern. Soc. Cell Biol., 7: 299-305, 1962. decreased cellular growth but promoted maturation. Ad- 22. Hay, E. D. Structure and Function of the Nucleolus in Developing Cells. In: J. Dalton and F. Haguenau (eds.). The Nucleus. New York: Academic amson and Klass (1) observed prednisolone to enhance Press. Inc.. 1968. maturation in culture of pulmonary adenoma cells into 23. Hogben. L. T. The Pigmentory Effector System: A Review of the Physi ology of Colour Response. London: Oliver and Boyd, 1924. cells morphologically indistinguishable from type II alveolar 24. Inoue, S. The Effect of Colchicine on the Microscopic and Submicros epithelial cells of the normal lung. copic Structure of the Mitotic Spindle Exptl. Cell Res. Suppl., 2: 305- Investigations of Inoue (24), Swann and Mitchison (53), 322,1952. 25. Inoue. S.. and Saito, H. Cell Mobility by Labile Association of Molecules. Eigsti and Dustin (14), Davidson ef al. (10), Brinkley ef al. The Nature of Mitotic Spindle Fibers and Their Role in Chromosome (4), and Inoue and Saito (25) showed that colchicine causes Movement. J. Gen. Physiol., 50 (Suppl.) 259-288. 1967 26. Jande, S. S. Fine Structure of Tadpole Melanophores. Anat. Record. metaphase arrest. This effect is thought to persist for about 754: 533-544, 1966. 15 hr, after which the cell either degenerates or passes 27. Jimbow, K., Pathak. M. R.. and Fitzpatrick, T. B Effect of Ultraviolet on through the rest of the cell cycle. In the present study the Distribution Pattern of Microfilaments and Microtubules and on the administration of colchicine to tumor cells of Harding-Pas Nucleus in Human Melanocytes. Yale J. Biol. Med., 46: 411-426, 1973. 28. Karlsson, J. O., and Stostrand, J. The Effect of Colchicine on Axonal sey melanoma induced the formation of microfilaments Transport of Protein in the Optic Nerve and Tract of the Rabbit. Brain that persist for at least as long as 10 weeks, the duration of Res., 73: 617-619, 1969. 29. Loader, K. R., and Nathaniel. E. J. H. Persistence of Colchicine Induced the study. 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Fig. 1. Electron micrograph of a melanoma cell in a nontreated tumor showing a highly irregular nucleus, a prominent nucleolus. and a peripheral aggregation of chromatin. The cytoplasm exhibits multiple Golgi complexes, polysomes. mitochondria, and melanin granules. * 8,500. Fig. ÃŒ.A conspicuous bundle of filaments (til) bordered by ribosomes in a cell of colchicine-treated tumor The presence of nuclear masses, some of which show penfiucioar space while others do not. suggests that this cell is in early prophase or latei telophase. Melanin granules serve as a marker and identify the cell as a melanorytn - 15,000 Fig. 3. Electron micrograph demonstrating the presence of microfilaments (fil) in cells of treated tumor generation 1 (2 weeks after cessation of colchicine treatment). Note the large numbers of virus particles, x 34,400. Fig. 4. Filaments in an interphase melanoma cell of treated tumor generation 2 (4 weeks after cessation of colchicine treatment). A conspicuous bundle of filaments (M) bordered by ribosomes and mitochondria is clearly visualized, x 25,000. Figs. 5 and 6. Electron micrographs demonstrating the presence of microfilaments (fil) in cells of treated tumor generation 3 (6 weeks after cessation of colchicine treatment). Fig. 5. x 38.000: Fig. 6, x 37,500. Fig. 7. Microfilaments permaating the cytoplasm of a cell in treated tumor generation 4 (8 weeks after cessation of colchicine treatment), x 28,725. Fig. 8. Electron micrograph illustrating a well-defined bundle of filaments (fil), perinuclear in position and bordered by polysomes. Note the well- developed Golgi complex (G) and melanin granules adjacent to it. Treated tumor generation 5 (10 weeks after cessation of colchicine treatment), x 22,575.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. Ultrastructural Study of the Persistence of Colchicine-induced Cytological Changes in Harding-Passey Melanoma

Kenneth R. Loader and Edward J. H. Nathaniel

Cancer Res 1978;38:168-176.

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