Cell cycle dependent changes in morphology

Printed in Sweden Copyright Q 1977 by Academic Press, Inc. Ail rights of reproduction in any form reserved ISSN 00144827

Experimental

CELL Studies

CYCLE

Cell Research 107 (1977) 89-94

DEPENDENT

with a Cold-sensitive

CHANGES Mutant

IN

of Chinese

MORPHOLOGY

Hamster

Ovary Cells

M. ST. J. CRANE,’ J. B. CLARKEZ and D. B. THOMAS’ ‘National Clinical

Institute Research

for Medical Research, Mill Hill, Centre, Northwick Park Hospital,

London Mid&.,

NW7 IAA, and HA1 3UJ, UK

SUMMARY cs4-D3, a cold-sensitive mutant of CHO cells is defective at the Gl interval of the cell cycle and exhibits a reversible change in morphology from a Bbroblast appearance to an “epithelial-like” form. Here, we have shown by time-lapse microcinematography that change in morphology, in either direction, is a post-mitotic event.

Changes in morphology of mammalian cells, in vitro, have often been correlated with altered growth characteristics [l-3]. “Normal” fibroblasts in culture are typically bipolar, spindle-shaped cells which grow in a controlled and organised manner. In contrast, virally-transformed cells have a characteristic irregular shape and exhibit a loss in growth control [4,5]. Several studies [l, 3,6,7,8] have indicated that the change in cell shape may be fundamental of the process of transformation. Puck and coworkers [9] have reported that Chinese hamster ovary cells, clone K, (CHO-K,), a typical transformed cell line, may undergo a “reverse transformation” after treatment with dibutyryl cyclic AMP (db-CAMP) and testosterone: CHO-K, are converted from a rounded form with surface “blebs” to a “normal fibroblast” shape with a smooth surface membrane. Several other features of a normal phenotype were re-established: reorganised growth pattern; increased collagen synthesis; decreased agglutinability

by plant lectins or specific antibody. Recently, we have described a cold-sensitive mutant of CHO cells, cs4-D3, which is defective at the Gl interval of the cell cycle [lo]: at the non-permissive temperature (33°C) cells undergo one round of division and accumulate at Gl. Moreover, there is a change in cell shape: during a temperature shift (3933°C) cells are converted from a spindle, fibroblast shape to an “epithelial” morphology and this change is reversible. Here, we have studied this event by timelapse microcinematography and show that change in shape, in either direction, is preceded by a round of cell division. METHODS

AND

MATERIALS

Cell culture Details for the isolation of clone c&D3 have been described ureviouslv fill. Wild-tvoe CHO cells were mutagen&d with ethyl *methane &lphonate and selected at 33°C bv the BUdR-visible light techniaue. Stock cultures were grown as monolayers in Falcon plastic culture flasks (surface area 25 cmp) at 39°C in Eagle’s basal diploid medium supplemented with 10 % Exprl

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Crane, Clarke

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Fig. I. Phase-contrast micrographs of clone cs4-D3 at 39°C. Coverslip cultures were fixed in ethanol/acetic acid (3: 1; v/v) for 10 min, rinsed three times in ab-

solute ethanol and air-dried. (a) Sparse; (h) confluent, cultures.

fetal calf serum, non-essential amino acids (Gibco B&cult Ltd) and antibiotics. They were maintained in a humidified atmosphere with 5% CO, and passaged twice weekly at a split ratio of 1: 20.

1540, Telemechanics Limited, Camberley, Surrey) maintained experimental cultures within +O.YC of the required temperature. Exposures were made using a Paillard Bolex H16 Reflex tine camera. fitted with an electric motor and controlled by the Paillard-Wild “variometer” time-lapse control equipment. The exposure time was fixed at 0.2 set and the intervals between exposures were set at 40 sec. Ilford Pan F negative film was used throughout. Experimental cultures were established in Falcon culture flasks and photoor 33+05"C. graphed for 45 h at either 39fO.W

Time-lapse microcinematography Time-lapse microcinematography was carried out with a Wild M40 inverted microscope (X 10 phase contrast objective) enclosed in a perspex cabinet fitted with cartridge heaters (Type No. CH2001, Heading Ltd, London). The temperature regulator (Type No. TC

Fig. 2. Phase-contrast micrographs of clone c&D3 at 33°C. Coverslip cultures were maintained at 33°C for (a) 2 days; (b) 10 days and fixed as above. ExptlCeNRes

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Cell cycle and cell shape

91

plateau value after 36 h (fig. 3). Since there was also a delay before growth arrest at 33°C it seemed possible that both events were interdependent. Time-lapse microcinematography Fig. 4a-h illustrates selected frames

Fig. 3. Abscissa: hours at non-permissive temwrature. 33°C; ordinate: (left) % cells, o-0, Elongate; 0-O; rounded; (right) % [sH]TdR-labelled nuclei (A-A). Change in morphology at 33°C. Coverslip cultures were shifted to 33°C and the number of elongate versus rounded cells were counted at zero time and various intervals thereafter, each point being a mean of three determinations. Triplicate samples were exposed to 0.2 &i rH]TdR for 30 min fixed in acetic acid/ ethanol (1: 3 v/v) and developed for autoradiography.

RESULTS Cell shape at 33°C and 39°C

At the permissive temperature (39°C) cs4D3 exhibits a typical fibroblast form (fig. la): cells are elongate and spindle-shaped with a smooth surface membrane and, at conlluency, cultures have an ordered and regular appearance (fig. 1b). Fig. 2 a illustrates the striking change in morphology of cultures maintained for 48 h at the nonpermissive temperature (33°C): cs4-D3 is converted from a fibroblast form to an “epithelial”, rounded shape with surface blebs. Cells are no longer arranged in parallel arrays and cultures lose their ordered appearance. After prolonged periods at 33°C (10 days, fig. 2b) a substantial number of cells are birefringent and resemble mitotic cells but are still firmly attached to the substratum. Change in morphology at the non-permissive temperature was not immediate and the proportion of “epithelial” cells reached a

taken from a time-lapse film of a cell culture shifted from 39°C to 33°C. At zero time (fig. 4a) the majority of cells (-90%) exhibited a “fibroblast” form, which was maintained through the intermitotic period. At mitosis, cells rounded up in the normal manner (fig. 4c, arrow I; fig. 4d, arrow I; fig. 4e, arrow II) but failed to spread out on the substratum following cytokinesis (fig. 4e-h, arrow I; fig. 4&h, arrow II). Cells remained rounded, showing active surface “blebbing”, yet were firmly attached to the substratum throughout the experiment. Cells that did not divide (fig. 4 a-h, arrow III) during this 45 h period remained elongate (Gl cells?). A delay of one cell cycle was also observed during a reverse shift from 33°C to 39°C: fig. Sa-h illustrates selected frames taken from a time-lapse film of a cell culture returned to the permissive temperature (39°C). At zero time most cells were in the rounded form and showed active surface “blebbing” (fig. 5a). Cells remained “epithelial-like” throughout the intermitotic period (18-20 h) and, at metaphase, resembled normal mitotic cells (fig. SC, arrow I; fig. Sf, arrow II). After cytokinesis, cells returned to their elongate form (fig. 5d, e , arrow I; fig. 5g, arrow II). DISCUSSION The results presented here indicate that change in morphology of clone cs4-D3 following a shift from permissive to non-permissive temperature (39°C to 33°C; fig. 4) or Exptt

Cell

Res 107 (1977)

4. Selected frames taken from a time-lapse microcinematograph of a cell culture stepped down from 39°C to 33fO.YC. (a) 0 h; (b) 3 h; (c) 7 h; (d) 7.5 h; (P) I1 h; (f) 14.5 h; (g) 21 h; (II) 40 h.

Fig.

the reverse (33°C to 39°C; fig. 5) is a postmitotic event. It is generally accepted that microtubules are responsible for the maintenance of shape in eukaryotic cells [12, 131: wild-type CHO cells, clone K1 are normally rounded in shape and may be converted to a “fibroblastic” form by addition of db-CAMP [6]. Puck et al. [9] have shown that the action of db-CAMP is antagonised by colcemid and cytochalasin B-agents that disrupt microExprl

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Res 107 (1977)

tubules and microfilaments, respectively. Furthermore, ultrastructural studies [12, 141 of CHO-K, and variants thereof have implicated microtubules in the control of cell shape. It was suggested that db-CAMP stimulates the polymerisation of the microtubular protein, tubulin, thereby increasing both the number and regular arrangement of the microtubules [ 151. The shape change induced in wild-type CHO-K, cells by db-CAMP is a cell cycle

dependent event: G’Neill and co-workers [16] showed that only Gl cells were responsive to db-CAMP. Our previously published data [lo] are consistent with these findings: at 33°C (non-permissive temperature) cs4-D3 is arrested at Gl with an altered morphology and can assume the elongate form within hours of treatment with db-c AMP. Microtubules have been implicated in the

regulation of cell growth [ 171. Brinkley et al. [18] have shown that transformed cells have fewer microtubules than their normal counterpart. Also, Vollet & Butel [19], using hamster fibroblasts infected with temperature-sensitive mutants of SV40, have reported that cells exhibit a typical transformed morphology under permissive conditions and resemble “normal” fibroblasts at the non-permissive temperature. If mi.fspf/Cd Rus107

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crotubules are indeed involved in the regulation of cell growth, clone cs4-D3 may offer a suitable model for their study. M. St. J. C. acknowledges Medical Research Council.

student support from the

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8. Johnson. G S. Friedman. R M 8z Pastan. 1. Proc natl acad sci US 68 (1971) 425. 9. Puck, T T, Waldren, C A & Hsie, A W, Proc natl acad sci US 69 (1972) 1943. 10. Crane. M St J & Thomas, D B, Nature 261 (1976) 205. 11. Farber, R & Unrau, P, Molec gen genet 138 (1975) 233. 12. Porter, K R, Puck, T T, Hsie, A W & Kelley, D, Cell 2 (1974) 145. 13. Tilney, L G & Porter, K R, J cell biol 34 (1967) 327. 14. Borman, L S, Dumont, J N L Hsie, A W, Exp cell res 91 (1975) 422. 15. Willingham, M C & Pastan, I, J cell bio167 (1975) 146. 16. O’Neill, J P, Schroder, C H, Riddle, J C & Hsie, A W, Exp cell res 97 (1976) 213. 17. Olsen, R W, J theor bio149 (1975) 263. 18. Brinkley, B R, Fuller, G M & Highfield, D P, Proc natl acad sci US 72 (1975) 498 1. 19. Vollet, J J & Butel, J S, J cell biol70 (1976) 51a. Received October 26, 1976 Accepted January 12, 1977