Brief Communication: Cilia Formation in Cultures of Human Lung Cancer Cells Treated With Dimethyl Sulfoxide 1 Tommie Sue Tralka 2 and Alan S. Rabson 3, 4 ABSTRACT-Cells from cultures of the ChaGo line of human lung cancer were examined periodically by electron microscopy for 4 years and no cilia were observed. However, when cells were grown in medium containing 2% dimethyl sulfoxide, as many as 50% of the cells had cilia with a 9+0 microtubular paHern.-J Natl Cancer Inst 57: 1383-1388, 1976.
MATERIALS AND METHODS
The ChaCo cells were grown in 75-cm 2 plastic flasks in medium composed of RPMI-1640 with 20% fetal bovine serum. The cells grew both adherent to the plastic surface and in suspension (3). In the initial experiment, one flask of ChaCo cells was fed with medium containing 2% DMSO whereas a control flask was kept in medium free of DMSO. After 18 days, the floating cells from each flask were collected, pelleted, and fixed for electron microscopy (4). Then half of each flask was scraped with a rubber policeman to remove adherent cells, which were pelleted and fixed for electron microscopy. The cultures were refed with DMSO-containing medium and control medium and 3 days later (21 days after initiation of the expt) processed for electron microscopy as above. RESULTS
(2)
(3)
Cilia were observed in 30-40% of the adherent cells after 18 days of DMSO treatment and in 40-50% after 21 days. No cilia were seen in the floating cells from treated or control cultures and in control adherent cells. In two other experiments with 2% DMSO, we found that the induction of cilia was reproducible. Cilia could be seen in a few cells after 7 days but could not be found in cultures after only 24 and 48 hours' exposure to 2% DMSO. The cilia usually were seen protruding into intraductular lumens within the cytoplasm (fig. 1), although they were occasionally seen at the cell periphery (fig. 2). All were of a 9+0 microtubular pattern when observed in cross-section (fig. 3). They varied in size from 1,500 to 2,200 nm in length and from 175 to 265 nm in diameter. Centrioles were often near the base of the cilia. Some VOL. 57, NO.6, DECEMBER 1976
REFERENCES (1) FRIEND C, SCHER W, HOLLAND JG, et al: Hemoglobin synthesis in
(4)
(5) (6)
1383
murine virus-induced leukemic cells in vitro. II. Stimulation of erythroid differentiation by dimethyl sulfoxide. Proc Natl Acad Sci USA 68:378-382, 1971 KIMHI Y, PALFREY C, SPECTOR I, et al: Maturation of neuroblastoma cells in the presence pf DMSO. Proc Natl Acad Sci USA 73:462-466, 1976 RABSON AS, ROSEN SW, TASHJIAN AH JR, et al: Production of human chorionic gonadotropin in vitro by a cell line derived from a carcinoma of the lung. J Natl Cancer Inst 50:669-674, 1973 RABSON AS, O'CONOR GT, LORENZ DE, et al: Lymphoid cell culture line derived from lymph node of marmoset infected with Herpesvirus saimiri-a preliminary report. J Natl Cancer Inst 46:1099-1109, 1971 GRILLO MA, PALAY SL: Ciliated Schwann cells in the autonomic nervous system of the adult rat. J Cell Bioi 16:430-436, 1963 ALLEN RA: Isolated cilia in inner retinal neurons and in retinal
ABBREVIATION USED: DMSO
=
dimethyl sulfoxide.
Received April I, 1976; accepted May 21,1976. Laboratory of Pathology, Division of Cancer Biology and Diagnosis, National Cancer Institute (NCI), National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 Division of Cancer Biology and Diagnosis, NCI. 4 We are grateful to Mr. E. R. Henson for his technical assistance. 1
2
J
NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
Friend et al. (1) made the important observation that the growth of Friend erythroleukemia cells in vitro in the presence of 2% DMSO markedly enhanced erythroid differentiation with an increase in hemoglobin synthesis. DMSO also enhanced differentiation of murine neuroblastoma cells with production of many long cellular processes (2). While sudying the effects of a number of substances known to induce oncogenic viruses in tumor cells, we found that 2% DMSO induced cilia formation in cultures of the ChaCo human lung cancer cell line (3).
cilia had a club-shaped free end (fig. 4), but most were pointed. With the exception of the cilia and seemingly fewer secretory type granules, the ChaGo cells remained morphologically the same as those previously observed. Most cells had large nuclei with prominent nucleoli. The cytoplasm contained abundant organelles including an active complex Golgi zone surrounded by small smooth endoplasmic reticulum vacuoles, moderate rough endoplasmic reticulum sometimes distended with an amorphous substance, and moderate to abundant mitochondria, some of which were large and bizarre. Underlying tonofibrils, microtubules, and free ribosomes were present. Glycogen, lysosomes, and lipid droplets were also observed. Occasionally, small dense granules thought to be secretory in nature were seen; more of these were observed in the controls. Desmosomes connected the cells. It is difficult to discuss the significance of induction of 9+0 cilia by DMSO, since the significance and function of these structures are not clear. Many types of differentiated mammalian cells possess 9+0 cilia (5-8), but 9+0 cilia can also be found in embryonic cells undergoing rapid division (9). The ability to induce 9+0 cilia in a high percentage of human lung cancer cells with DMSO may make it possible to examine in more detail the question of their role in growth control and differentiation of malignant cells.
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pigment epithelium. J Ultrastruct Res 12:730-747, 1965 (7) SOROKIN S: Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Bioi 15:363-377 , 1962 (8) - - - : Single, primary or oligocilia. In Ultrastructural Pathology of the Cell. A Text and Atlas of Physiological and Pathological
Alteration in Cell Fine Structure (Ghadially F, ed.). London, Boston, Butterworth, 1975, pp 512-519 (9) FONTE VG, SEARLS RL, HILFER SR: The relationship of cilia with cell division and differentiation. J Cell Bioi 49:226-229, 1971
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
J
NATL CANCER INST
VOL. 57, NO.6, DECEMBER 1976
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
I.-Longitudinal section of a cilium (C) extending into a ductular lumen in cytoplasm of a ChaGo cell that had been incubated 7 days at 35 0 C in medium containing 2% DMSO. x 34,000
FIGURE
TRALKA AND RABSON
1385
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
2.-Longitudinal section of cilium within pouch at cell periphery. Tubules extend from distal centriole (C,) through shaft up to a bend of appproximately 45 degrees and reappear in end of shaft (S) which is cut at a different level. Proximal centriole (C 2 ) is shown at a tangential level. Cells were incubated 18 days in media containing 2% DMSO. X 34,000
FIGURE
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TRALKA AND RABSON
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
3.-Cross-section of cilium (C l ) within cytoplasmic vacuole after 18 days in media containing 2% DMSO. It shows 9 outer microtubule doublets with absence of central tubules. Two centrioles (C 2 , C3 ) are near, with satellites (arrows) between C 2 and C l • x 114,000
FIGURE
TRALKA AND RABSON
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Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
4.-Longitudinal section of bulbous-ended cilium (C) projecting into ductular lumen incubation in medium containing 2% DMSO. X 34,000
FIGURE
1388
In
cytoplasm of a ChaGo cell after 21 days'
TRALKA AND RABSON
MATERIALS AND METHODS
The ChaCo cells were grown in 75-cm 2 plastic flasks in medium composed of RPMI-1640 with 20% fetal bovine serum. The cells grew both adherent to the plastic surface and in suspension (3). In the initial experiment, one flask of ChaCo cells was fed with medium containing 2% DMSO whereas a control flask was kept in medium free of DMSO. After 18 days, the floating cells from each flask were collected, pelleted, and fixed for electron microscopy (4). Then half of each flask was scraped with a rubber policeman to remove adherent cells, which were pelleted and fixed for electron microscopy. The cultures were refed with DMSO-containing medium and control medium and 3 days later (21 days after initiation of the expt) processed for electron microscopy as above. RESULTS
(2)
(3)
Cilia were observed in 30-40% of the adherent cells after 18 days of DMSO treatment and in 40-50% after 21 days. No cilia were seen in the floating cells from treated or control cultures and in control adherent cells. In two other experiments with 2% DMSO, we found that the induction of cilia was reproducible. Cilia could be seen in a few cells after 7 days but could not be found in cultures after only 24 and 48 hours' exposure to 2% DMSO. The cilia usually were seen protruding into intraductular lumens within the cytoplasm (fig. 1), although they were occasionally seen at the cell periphery (fig. 2). All were of a 9+0 microtubular pattern when observed in cross-section (fig. 3). They varied in size from 1,500 to 2,200 nm in length and from 175 to 265 nm in diameter. Centrioles were often near the base of the cilia. Some VOL. 57, NO.6, DECEMBER 1976
REFERENCES (1) FRIEND C, SCHER W, HOLLAND JG, et al: Hemoglobin synthesis in
(4)
(5) (6)
1383
murine virus-induced leukemic cells in vitro. II. Stimulation of erythroid differentiation by dimethyl sulfoxide. Proc Natl Acad Sci USA 68:378-382, 1971 KIMHI Y, PALFREY C, SPECTOR I, et al: Maturation of neuroblastoma cells in the presence pf DMSO. Proc Natl Acad Sci USA 73:462-466, 1976 RABSON AS, ROSEN SW, TASHJIAN AH JR, et al: Production of human chorionic gonadotropin in vitro by a cell line derived from a carcinoma of the lung. J Natl Cancer Inst 50:669-674, 1973 RABSON AS, O'CONOR GT, LORENZ DE, et al: Lymphoid cell culture line derived from lymph node of marmoset infected with Herpesvirus saimiri-a preliminary report. J Natl Cancer Inst 46:1099-1109, 1971 GRILLO MA, PALAY SL: Ciliated Schwann cells in the autonomic nervous system of the adult rat. J Cell Bioi 16:430-436, 1963 ALLEN RA: Isolated cilia in inner retinal neurons and in retinal
ABBREVIATION USED: DMSO
=
dimethyl sulfoxide.
Received April I, 1976; accepted May 21,1976. Laboratory of Pathology, Division of Cancer Biology and Diagnosis, National Cancer Institute (NCI), National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 Division of Cancer Biology and Diagnosis, NCI. 4 We are grateful to Mr. E. R. Henson for his technical assistance. 1
2
J
NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
Friend et al. (1) made the important observation that the growth of Friend erythroleukemia cells in vitro in the presence of 2% DMSO markedly enhanced erythroid differentiation with an increase in hemoglobin synthesis. DMSO also enhanced differentiation of murine neuroblastoma cells with production of many long cellular processes (2). While sudying the effects of a number of substances known to induce oncogenic viruses in tumor cells, we found that 2% DMSO induced cilia formation in cultures of the ChaCo human lung cancer cell line (3).
cilia had a club-shaped free end (fig. 4), but most were pointed. With the exception of the cilia and seemingly fewer secretory type granules, the ChaGo cells remained morphologically the same as those previously observed. Most cells had large nuclei with prominent nucleoli. The cytoplasm contained abundant organelles including an active complex Golgi zone surrounded by small smooth endoplasmic reticulum vacuoles, moderate rough endoplasmic reticulum sometimes distended with an amorphous substance, and moderate to abundant mitochondria, some of which were large and bizarre. Underlying tonofibrils, microtubules, and free ribosomes were present. Glycogen, lysosomes, and lipid droplets were also observed. Occasionally, small dense granules thought to be secretory in nature were seen; more of these were observed in the controls. Desmosomes connected the cells. It is difficult to discuss the significance of induction of 9+0 cilia by DMSO, since the significance and function of these structures are not clear. Many types of differentiated mammalian cells possess 9+0 cilia (5-8), but 9+0 cilia can also be found in embryonic cells undergoing rapid division (9). The ability to induce 9+0 cilia in a high percentage of human lung cancer cells with DMSO may make it possible to examine in more detail the question of their role in growth control and differentiation of malignant cells.
1384
TRALKA AND RABSON
pigment epithelium. J Ultrastruct Res 12:730-747, 1965 (7) SOROKIN S: Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Bioi 15:363-377 , 1962 (8) - - - : Single, primary or oligocilia. In Ultrastructural Pathology of the Cell. A Text and Atlas of Physiological and Pathological
Alteration in Cell Fine Structure (Ghadially F, ed.). London, Boston, Butterworth, 1975, pp 512-519 (9) FONTE VG, SEARLS RL, HILFER SR: The relationship of cilia with cell division and differentiation. J Cell Bioi 49:226-229, 1971
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
J
NATL CANCER INST
VOL. 57, NO.6, DECEMBER 1976
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
I.-Longitudinal section of a cilium (C) extending into a ductular lumen in cytoplasm of a ChaGo cell that had been incubated 7 days at 35 0 C in medium containing 2% DMSO. x 34,000
FIGURE
TRALKA AND RABSON
1385
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
2.-Longitudinal section of cilium within pouch at cell periphery. Tubules extend from distal centriole (C,) through shaft up to a bend of appproximately 45 degrees and reappear in end of shaft (S) which is cut at a different level. Proximal centriole (C 2 ) is shown at a tangential level. Cells were incubated 18 days in media containing 2% DMSO. X 34,000
FIGURE
1386
TRALKA AND RABSON
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
3.-Cross-section of cilium (C l ) within cytoplasmic vacuole after 18 days in media containing 2% DMSO. It shows 9 outer microtubule doublets with absence of central tubules. Two centrioles (C 2 , C3 ) are near, with satellites (arrows) between C 2 and C l • x 114,000
FIGURE
TRALKA AND RABSON
1387
Downloaded from http://jnci.oxfordjournals.org/ at University of Otago on October 21, 2015
4.-Longitudinal section of bulbous-ended cilium (C) projecting into ductular lumen incubation in medium containing 2% DMSO. X 34,000
FIGURE
1388
In
cytoplasm of a ChaGo cell after 21 days'
TRALKA AND RABSON
