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Electron Microscope Studies on HeLa Cell Lines Sensitive and Resistant to Actinomycin D* L. J. JOURNEY AND MILTON N . GOLDSTEIN (Department of Experimental Biology, Roswell Park Memorial Institute, Buffalo, New York) SUMS{ARY The fine structure of stock IIeLa cells has been examined, and several new details have been described. Certain specific structural alterations induced by actinomycin D, a cytotoxic antibiotic, have been noted. These alterations included cytoplasmic blebbing and a fragmentation of the nucleoli accompanied by a pronounced loss in their osmiophilia, tteLa cells made resistant to actinomycin D did not display these structural changes. The findings are discussed in the light of histochemical and biochemical evidence which indicates that actinomycin D inhibits RNA and protein synthesis. Actinomycin D, an antibiotic isolated by Manaker et al. (19) from Streptomyces parvallus, has caused regression of some animal and human tumors ( l l , 22, 25). This antibiotic is toxic in vivo, and cytotoxic effects are produced in cells of newly explanted and established cell lines grown in vitro (4, 6, 7, 12). Recently, we reported some cytological, histochemical, and chemical studies on a stock HeLa line which was sensitive to actinomycin D and a resistant line isolated from the stock HeLa cells (15). The drug produced alterations in nucleolar structure and a decrease in staining intensity for R N A in nucleoli and cytoplasm of sensitive cells, whereas the resistant cells were not affected. Chemical assays revealed a decrease in R N A and protein, whereas D N A was unchanged in treated, sensitive cells. The present report describes more detailed phase and electron microscopic observations on sensitive HeLa cells treated with actinomycin D and a HeLa line made resistant to the drug. 199. Details of culture technics and development of the resistant line are described elsewhere (]5). The experimental material included three cell strains: 1. HeLa-S--stock cell line that was sensitive to actinomycin D. 2. H e L a - S R - - a partially resistant sublinc selected from the above stock culture by intermittent exposure to actinomycin D. These cells survived for longer than 10 days in 0.1 #g/ml actinomycin D, a concentration lethal for sensitive cells within 72 hours. S. H e L a - R - - a resistant subline that can actively multiply in medium containing 0.1 pg/ml of the antibiotic. For E M studies the cells were explanted into 15 X 150 ram. :Pyrex test tubes containing 1.5 ml. of medium. Cultures were refed with experimental medium 24 hours after explantation. At the end of the experimental period (24-48 hours), controls and drug-exposed cells were washed in Hanks solution and fixed for 10 minutes in osmium, vapor. The cells were then rapidly dehydrated in a graded series of alcohols. Final embedding was M A T E R I A L S AND M E T H O D S done in pre-polymerized methacrylate (4 butyl-IThe HeLa lines were derived from stock HeLa 1 methyl) and polymerization completed at cells that have been serially propagated in our 70~ C. Embedded cells were removed by cracking laboratory for the past 5 years in a medium com- the tubes in a freezing mixture of dry ice and alcoposed of 1 part calf serum and 2 parts of medium hol. Selected areas were cut out, oriented, cemented to Lucite rods, and sectioned in a :Porter* Supported in part by research grant t/Dl~G-527 from tlle Blum microtome. The sections were picked up on Damon Runyon Memorial Fund for Cancer l~esearch. Formvar-coated grids and examined in an RCA Received for publication March 9, 1.q6l. EMU-2B microscope. 929 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. 930 Cancer Research RESULTS Stock HeLa.--When explanted in roller tubes, cells adhered to the glass and grew out in dense monolayers. Cell surfaces had numerous villi, which interdigitated with surrounding cells (Figs. 3 and 7). In certain areas cell surfaces were in close apposition, but in other portions of the sheet cells were separated by wide intercellular spaces. Typically, each control cell possessed a large ovoid nucleus, though in some dense areas multinucleated cells were apparent. Some nuclei had deep invaginations, and others had bhmt protrusions or lobulations. The nucleoplasm was composed of fine, granular material distributed homogeneously. Chromatin material was evenly disperscd throughout the nucleoplasm with no margination along the nuclear envelope. All stages of mitosis wcre cncountered; chromosomes appeared as condensed masses of densely staining granules, and the filamentous structure of the spindle apparatus was evident. Nuclei contained one to five nucleoli, which varied considerably in size, and many were found adjacent to the nuclear envelope. Some nucleolar structures appeared as profiles of coiled threadlike nucleolonema (Fig. 7), and a few contained dense inclusions. At higher magnifications, nucleoli presented the typical picture of osmiophilic granules resembing the ribonucleoprotein particles of the cytoplasm. Mitochondria were extremely pleomorphic, ranging from small, round types to huge elongate varieties, which may branch considerably to produce bizarre shapes (Fig. 3). The arrangement of internal cristae was variable. Septation was transverse, oblique, or longitudinal with respect to the long axis of the mitochondrion. Golgi material was usually found near the nucleus and consisted of a complex network of membranes with associated vacuoles and granules (Fig. 7). Dense bodies, smaller than mitochondria and with no internal structure, were also present (Fig. 7). These structures resembled the "globoid bodies" found in HeLa cells by Harford et al. (16). Similar granules have been described in other tissues and have been termed "microbodies" (24), "peribiliary bodies" (21), "dense bodies" (20), and apparently correspond to the "lysosome" fraction obtained from tissue homogenates (5). Organized cndoplasmic reticulum was sparse in HeLa cells, with little evidence of the orderly array of tubules and sacs seen in many normal tissues. This systcm was represented predominantly as isolated vesicles and tubules dispersed throughout thc cytoplasm. Other cytoplasmic structures found occasionally were areas composed Vol. ~1, A u g u s t 1961 of fibrous material. In many cells, certain areas of the cytoplasm appeared hyaline or less osmiophilic than surrounding portions (Fig. 7). Although sharply demarcated, these areas were not membrane-enclosed and contained the same granular background as adjacent portions of the cytoplasm. HeLa-S + Actinomycin D.--After exposure to 1 gg/ml of actinomycin D for 20 hours, extensive cytotoxic alterations were evident in sensitive HeLa cells. Some cells showed cytoplasmic blebbing, i.e., out-pocketing and pinching-off of cytoplasmic contents. This process is demonstrated in Figure 2, a phase-contrast picture of a living HeLa cell with numerous surface blebs. A HeLa cell in control medium (Fig. 1) is included for comparison. Successive stages in the formation and release of cytoplasmic blebs are seen in greater detail with electron microscopy (Fig. 6). Other alterations in fine structure inchlded vacuolization of mitochondria and crgastoplasmic vesicles, and disruption of nuclear contents. Nucleoli were especially sensitive to the cytotoxic effects of actinomycin D. They were converted from densely stained bodies to nucleolar structures composed of a hyaline, washed-out interior enclosed in a peripheral ring of osmiophilic granules. Actinomycin D also induced a marked budding and fragmentation of the nucleoli (Figs. 5 and 6). I t should bc emphasized that nucleolar alterations were apparent in all of tile cells examined. HeLa-SR.--The following observations were made on a subline intermittently exposed to actinomycin D and fixed for electron microscopic examination after 24 hours in medium free of the drug. In general, the structure of these partially resistant cells was unchanged. Some mitochondria were swollen b u t were often found in close proximity to mitochondria of normal structure. Nucleoli resembled those of the untreated sensitive line (Fig. 7). Large nuclear inclusions, not seen in control cells, were present in a few cells. Phagocytosis was more common, undoubtedly due to the increased number of dead cells in the cultures. When these partially resistant cells were fixed while still in media containing actinomycin D, nucleolar alterations similar to those seen in sensitive cells were invariably present. Nucleoli appeared as small, round bodies with structureless interiors, the majority of which were in the process of division or budding. Nucleolar fragmentation led to the appearance of many small round nucleoli. HeLa-R.--There were no apparent morphologic differences between resistant cells growing in normal medium or those growing in medium con- Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. JOURNEY AND GOLDSTEIN--HeLa Cells and Actinomycin D raining 0.1 gg/ml of the drug. In general, resistant cells were smaller, with less cytoplasm than sensitive HeLa cells. Occasionally, cells differing from the typical epithelial type were found; these variants were long, spindle-shaped cells and were usually isolated from other cells. There was no evidence of vesiculation of cell surfaces as seen in sensitive cells. The most striking difference in resistant cells was the appearance of the nucleolus. Nucleoli of resistant cells were large and dense as compared with the small, hyaline type found in drug-treated sensitive cells (Fig. 8). M a n y nuclei were lobulated and infolded and in some instances contained membrane-bound inclusions. Cytoplasmic organelles were similar to those of control cells, although vacuolization was common. Phagocytic vacuoles were frequently present, either as large, empty vacuoles or numerous small secondary masses scattered through the cytoplasm. DISCUSSION The principal cytotoxic effects in sensitive HeLa cells treated with actinomycin D were: (a) nucleolar alterations, and (b) blebbing and pinching-off of cytoplasm from the cell surface. Nucleolar changes were observed consistently in sensitive and partially resistant HeLa cells in the presence of very low concentrations (0.1 to 0.001 #g/ml) and appear to be a specific effect of actinomycin D. Fragmentation or budding of nucleoli and conversion to the lightly stained, hyaline type were invariably present in drug-treated cells. These observations confirmed and extended our previous light microscope findings with histological and histochemical staining procedures. In general, nucleolar morphology is labile and responsive to variations in the metabolic activity of cells. Nucleoli are hypertrophied in embryonic and other cells displaying active synthesis and reduced in quiescent cells. Structural changes can also be produced by a wide variety of physical and chemical agents (~6). For example, Hughes (17) noted that several purine derivatives caused fragmentation of nucleoli in chick tissue cultures, which reconstituted when cells were washed free of drug. In this regard, most sensitive HeLa cells quickly recovered when rinsed free of highly cytotoxic concentrations of actinomycin D. Suggestive evidence for the mechanism underlying altered staining affinity of nucleoli in drugtreated cells was obtained by chemical assays for RNA, DNA, and protein. Preliminary experiments revealed that both R N A and protein, the primary constitutents of nucleoli, were depleted in sensitive cells, whereas D N A content was unchanged (15). 931 Blebbing of the cell membranes suggested that actinomycin D may act primarily at the cell surface. I t should be noted, however, that these drastic surface changes were observed only occasionally. Although treatment with antibody produced vesiculation of cell membranes in ascites tumor cells (14) and HeLa cells, 1 these vesicles were essentially devoid of cytoplasmic contents. There is no available information on the site of action of actinomycin D. To examine this problem, experiments with labeled actinomycin D are planned to study its permeability in HeLa cells and possible sites of intracellular accumulation. Additional morphological features not previously described in HeLa strains were hyaline or less osmiophilic regions and fibrous areas in the cytoplasm of some cells. These areas were not due to actinomycin D, since they were found in control as well as treated cell lines. Edwards and Fogh (8) described structural alterations in normal human amnion cultures produced by long exposure to trypsin. Among these alterations were areas of fine filaments and hyaline cytoplasmic regions, although no differentiation was made between these two dissimilar entities. Although brief exposure to trypsin was used routinely in subculturing our HeLa lines, hyaline regions were not a consistent finding. Recent evidence (~3) indicates that these hyaline regions probably represent areas of glycogen deposition. The presence of concentrically arranged fibrillar areas has been noted in leukemic blast cells (3, 13), solid (~, 9), and ascites tumor cells (1, 10). Similar fibrils were found in our HeLa lines. These fibrous components were more prevalent in resistant HeLa cells, but their origin o r significance is uncertain. The most remarkable cytological feature of fully resistant HeLa cells was the persistence of prominent, intensely osmiophilic nucleoli with no evidence of fragmentation. There are several possible mechanisms by which cells might become resistant to actinomycin D: (a) cell surface changes prohibit entry of the drug; (b) the drug is bound or detoxified within the cell; and (c) induction of a hydrolytic enzyme to inactivate the drug. In this regard Katz and Pienta (18) have presented evidence which indicated that an enzyme system is involved in the decomposition of actinomycin by bacterial organisms. There is, at present, no evidence to indicate that higher organisms possess a similar enzyme system. The function of the nucleolus is incompletely understood. Since actinomycin D appears to have a specific effect on nucleolar morphology, it should 1Unpublished observations. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. 93~ Cancer Research prove useful in exploring the role of this structure in cell activities. ACKNOWLEDGMENTS The authors wish to acknowledge the assistance of Eva Havas Pfendt and Charlotte Pollard. REFERENCES 1. BERGSTRASD,A., and RINGERTZ, N. Electron Microscopic Examination of the MC1M Tumor. I. The Tumor in Ascites Form. J. Nat. Cancer Inst., 2fi:501-21, 1960. ~. BERNHARD, W., and de HARVEN, E. Sur la pr6sence dana certaines cellules de Mammif~res d'un organit6 de nature probablement centriolaire. ]~tude au microscope 61ectronlque. Compt. rend. Acad. Sc., 242:~88-90, 1956. 3. BESSIS, M., and BRETON-GOR~US, J. Sur les formations particuli~res observdes au microscope 61ectronique dans certaines cellules leuc6miques. Compt. rend. Acad. Sc., 240: 459-60, 1955. 4. COBB, J. P., and WALKER, D. Effect of actinomycin D on Tissue Cultures of Normal and Neoplastic Cells. J. Nat. Cancer Inst., 21: 06.'3-77, 1958. 5. DE DUVE, C. Lysosomes, a New Group of Cytoplasmic Particles. Its: T. HAYASIII (ed.), Subcellular Particles, pp. 10-8-60. New York: Ronald Press Co., 1959. 6. EAGLE, II., and FOLEY, G. E. The Cytotoxic Action of Carcinolytic Agents in Tissue Culture. Am. J. Med., 21:73949, 1956. 7. ~ . Cytotoxicity in Human Cell Cultures as a Primary Screen for the Detection of Antitumor Agents. Cancer Research, 18:1017-110-5, 1958. 8. EDWARDS,G. E., and FoGH, J. Micromorphologic Changes in Human Amnion Cells during Trypsinization. Cancer Research, 19: 608-11, 1959. 9. EDWARDS,G. E.; R(rSKA, C.; RUSK)., II.; and SKIFF, J. V. The Micromorphology of a Human Bronchogenic Carcinoma. Cancer, 12:980--100~, 1959. 10. EPSTEIN, M. A. The Fine Structure of the Cells in Mouse Sarcoma 37 Ascitic Fluid. J. Biophys. & Biochem. Cytol., 3: 567-76, 1957. 11. FARBER, S. Carcinolytic Action of Antibiotics, Puromycin and Actinomycin D. Am. J. Path., 31:580-86, 1955. 1~. FOLEY, G. E., and EAGLE, H. The Cytotoxicity of Antitumor Agents for Normal Human and Animal Cells in First Tissue Culture Passage. Cancer Research, 18:101~16, 1958. Fio. 1.--Living stock HeLa cell in normal medium. Note the villi along the limiting boundary of the cell. Long filamentons mitochondria are scattered through the cytoplasm, and dense lipide droplets are concentrated near the nucleus. The nucleoli, which are out of focus, appear as dense ovoid bodies. Positive phase contrast, )<1,600. Vol. ~1, August 1961 13. FREE:~AN, J. A., and SA~IUEr~, M. The Ultrastructure of a "Fibrillar Formation" of Leukemic Hiiman Blood. Blood, 13: 7~5-0-8, 1958. 14. GOLDBERG, G., and GREEN, H. J. The Cytotoxic Action of Immune Gamma Globulin and Complement on Krebs Ascites Tumor Cells. I. Ultrastructural Studies. J. Exper. Med., 109: 505-10, 1959. 15. GOLDSTEIN, M. N.; SLOTNICK, I. J.; and JOURNEY, L. J. I n Vitro Studies with HeLa Cell Lines Sensitive and Resistant to Actinomycin D. Ann. New York Acad. Sc., 89: 474-83, 1960. 16. HARFORD, C. G.; HAMLIN, A.; and PARKER, E. Electron Microscopy of HeLa Cells after Ingestion of Colloidal Gold. J. Biophys. & Biochem. Cytol., 3:749-56, 1959. 17. HUOHES, A. F. The Effect of Purines and Related Substances upon Cells in Chick Tissue Cultures. Exper. Cell Research, 3:108-0-0, 195o.. 18. KxTz, E., and PIENTA, P. Decomposition of Actinomycin by a Soil Organism. Science, 126:400-3, 1957. 19. MANAKER, R. A.; GREGORY, F. J.; VINING, L. C.; and WAKS.~AN, S. A. Actinomycin III. The Production and Properties of a New Actinomycin. In: Antibiotics Annual, pp. 853-57. New York: Medical Encyclopedia, Inc., 19541955. ~0. NOV1KOFF,A. B.; BEAUFAY, H.; and DE DIyVE, C. Electron Microscopy of Lysosome-rich Fractions from Rat Liver. J. Biophys. & Biochem. Cytol. 2 (Suppl.): 179-84, 1956. ~1. PALADE, G. E., and SIEKEVITZ, P. Liver Microsomcs. An Integrated Morphological and Biochemical Study. J. Biophys. & Biochem. Cytol., 2:171-o00, 1956. ~0-. PINKEL, D. Actinomycin D in Childhood Cancer. Pediatrics, 23: 340--47, 1959. 0-3. REVEL, J. P.; NAPOLITANO, L.; and FAWCETT, 1). W. Identification of Glycogen in Electron Micrographs of Thin Tissue Suctions. J. Biophys. & Biochem. Cytol., 8: 575-91, 1960. ~4. ROUILLER, C., and BERNUARD, W. "Microbodies" and the Problem of 1VIitochondrial Regeneration in Liver Cells. J. Biophys. & Biochem. Cytol., 2:355-61, 1956. 0.5. SUGIUaA, R. Studies in a Tumor Spectrum. VIII. The Effect of Mitomycin C on the Growth of a Variety of Mouse, Rat, and lIamster Tumors. Cancer Research, 19: 438-45, 1959. 0.6. VINCENT, W. S. Structure and Chemistry of Nuclcoli. Internat. Rev. Cytol., 4:069-98, 1955. FrG. 0.--Living stock HeLa cell incubated for 04 hours in medium containing 0.1 gg/ml of actinomycin D. Note the irregular ruffled contour of the cell boundary. There are numerous blebs and cytoplasmic droplets (arrows) which have broken away from the cell surface. The nucleoli have a mulberry-like configuration and appear smaller than those in control cells. Positive phase contrast, X0-,000. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. i Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. FIG. 3.--Stock HeLa cell containing three dense nucleoli. Numerous pleomorphic mitochondria (some appear vacuolated) and several tubules of tile endoplasmic rcticulum (ER) are found in this section. In lower portion are profiles of microvilli which project from surfaces of the neighboring cells. X 1S,O00. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. i Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. ~IG. ~.--Micrograph showing nucleolar changes in stock HeLa cells after treatment with actinomycin D. The hyaline interior arid dense peripheral granules are clearly evident, and two buds have formed. X 13,000. FIG 5.--Another variation of nucleolar fragmentation produced in stock HeLa cells by actinomycin 1). X 16,000. FIG. 6.--Stock HeLa cell after exposure to 1 #g/ml actinomycin D. Cytoplasmic blebs in various stages of formation and release are seen. Mitochondria are vacuolated, and nucleoplasm appears disorganized. Dense granular accumulations in nucleus (N) m a y be either chromatin or remI~ants of disintegrated nucleoli. X7,000. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. i Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. FIG. 7.--Micrograph of partially resistant HeLa cells fixed after ~4 hours in medium free of actinomycin D. Nucleolus is densely stained; two nuclear indentations (ID) are indicated. Several "lysosomal" granules (LY) and the Golgi complex (G) are present. A hyaline area (HA) occupies a portion of the cytoplasm in each cell. >(13,000. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. l Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. FIG. 8.--Micrograph of resistant HeLa cells, i.e., ceils that grow and are maintained in media containing 0.1 ~g/ml actinomycin D. Nucleoli of resistant cells appear dense and intensely osmiophilic. X7,000. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. i i Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research. Electron Microscope Studies on HeLa Cell Lines Sensitive and Resistant to Actinomycin D L. J. Journey and Milton N. Goldstein Cancer Res 1961;21:929. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/21/7/929 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer Research.