The cell cycle of Xenopus laevis cells in monolayer culture

Preliminary notes 433 The results presented above indicate that human and mouse chromosomes can easily be distinguished in human-mouse hybrids following growth in BUdR and staining with Hoechst. Other 33258 techniques have been used to distinguish between human and mouse chromosomes in hybrid cells. However, with QM banding [7] and also with centric heterochromatin staining [lo], it is sometimes difficult to distinguish some of the small human chromosomes from mouse acrocentric chromosomes. Since all of the chromosomes in RAG cells show a half fluorescent centromere under the conditions described above, whereas all of the human chromosomes have a dull centromere, the technique described in this paper can facilitate the identification of the human chromosomes in humanmouse hybrids. (The identification of chromosomes according to centromere staining assumes that no recombination has occurred between human and mouse chromosomes.) The technique can also be used for rapidly determining the number of human chromosomes in human-mouse hybrids without the necessity of specifically identifying each chromosome. The occurrence of half fluorescent centromeres after BUdR-Hoechst treatment has been observed in all other cells of mouse origin (Mars muscuh) examined. This includes cells of three other permanent lines3T3, A9, and NCTC 2472-and also cells of a diploid strain of fibroblasts. It has recently been observed that the centromeric regions of the chromosomes of two other rodent species, Chinese hamster and Syrian hamster, exhibit a dull centromere after BUdRHoechst treatment, unlike the half-fluorescent centromere in the mouse (Lin & Davidson, unpublished). Thus the technique described in this paper can be used to indicate the species of origin of the chromosomes in hamstermouse, as well as human-mouse, hybrids.

This work was supported by grants from the National Institute of Child Health and Human Development (HD-06276 and HD-04807) and the National Institute of General Medical Sciences (GM-21 121).

References 1. Davidson, R L E &de la Cruz, F F (ed), Somatic cell hybridization, 295 pp. Raven Press, New York (1974). 2. Weiss, M. & Green, H, Proc natl acad sci US 58(1967) 1104. 3. Latt. S A. Proc natl acad sci US 70(1973) 3395. 4. Lin,‘M S: Latt, S A & Davidson, k L; Exptl cell res 86(1974) 392. 5. Littlefield, J, Science 145(1964) 709. 6. Davidson, R L & Ephrussi, B, Nature 205(1965) 1170. 7. Casoersson. T. Zech. L. Harris. H. Wiener, F & Klem, G, Exptl cell res’65(1971) 475. 8. Hilwig, I & Gropp, A, Exptl cell res 75(1972) 122. 9. Flamm, W G, McCallum, M & Walker, P, Proc natl acad sci US 57(1967) 1729. 10. Chen, T R & Ruddle, F H, Chromosoma 34(1971) 51. Received April 3, 1974 Revised version received May 6, 1974

The cell cycle of Xenopus Zuevis cells in monolayer culture P. M. GODSELL, School of Biological Universiiy of East Anglia, Norwich, UK

Sciences,

Summary. Analysis of the cell cycle of recently isolated Xenopus Zaevis cells in culture gave mean values for the duration of Gl, S and G2 of 18.0, 8.2 and 5.3 h respectively. After 70 weekly subcultures, cells with 38 chromosomes had equivalent values of 4.7, 6.3 and 3.0 h, and for tetraploid cells of 7.0,8.9 and 5.0 h. The duration of mitosis was 1.0 h.

The cell cycle of mammalian cells, both in vivo and in vitro has been sufficiently well characterised for Mitchison [l] to express the results of over 60 papers in histogram form, showing that for mammalian cells DNA synthesis normally lasts between 6 and 12 h, and that the greatest variations occur in the duration of G 1. In contrast, few measurements of amphibian cell cycle phases have been made, especially for cells in vitro, but the values obtained [2-71 suggest that the cell cycle of amphibia is generally longer than that of mammals, with the total cycle Exptl Cell Res 87 (1974)

434

Preliminary notes

Table 1. Cell cycle duration, labelled fraction and duration of the cell cycle phases of estabIised Xenopus laevis cells

Cell line

EAX 09/75 EAX 11175

Cell cycle time (hours)

% labelled cells

No. of subcultures

Uncorrected

Corrected

Uncorrected

Corrected

GI

S

G2

ma

17.1

14.5

35.0

42.1

4.7

6.3

3.0

15” 7oc

19.0 22.9

17.1 21.4

30.0 36.5

33.4 39.1

7.7 7.0

6.2 8.9

2.7 5.0

75d

19.4

Il.7

41.5

45.6

4.5

8.2

4.5

Duration of phase (hours)

% viability: ‘85.0; “90.0; ‘93.3; d91.0. The duration of mitosis was assumed to be 0.5 h in all cases.

normally 40 to 70 h, Gl from 0 to 40 h, DNA synthesis between 20 and 50 h and G2 up to 10 h in duration. However, Xenopus laevis cells are capable of relatively rapid proliferation. Measurements on cells in culture showed that population doubling times of 24 h or less were common [8, 91. These values, which are similar to those of many established cell lines, indicate that rapid amphibian cell proliferation is possible despite the lower incubation temperature. To determine the duration of the cell cycle phases of Xenopus laevis, measurements were made on both recently isolated and established cell lines. Materials and Methods Culture methods and other details of the cell lines used have been described [9]. Established cell lines EAX 09/75 (38 chromosomes) and EAX 11/75 (approximately tetraploid) subcultured at weekly intervals and growing exponentially at 25°C were used. Cell cycle parameters were estimated using the methods of Watanabe & Okada [lo] and Okada [ll] with slight modifications. The duration of mitosis (TM) was determined by direct observation of established kidney cells growing on coverglasses. Observations were made in a constant temperature room at 25°C. Coverglasses were inverted over a medium-filled cavity made with silicone adhesive on a microscope slide. Eleven cells were timed in each of three cell lines. The duration of G2 ( 7’02) was measured by following the appearance of labelled metaphase cells in autoradiographs of cultures continuously exposed to 3H-thymidine. Cells growing Exptl Cell Res 87 (I974

on coverglasses were fixed in methanol-acetic acid fixative at 2 h intervals after the addition of 1 ,&i/ml 3H-thymidine (Radiochemical Centre). After coating with Ilford K2 nuclear research emulsion the autoradiographs were exposed for 30 to 60 days and then lightly stained with Giemsa. The proportion of 50 metaphase cells labelled was determined for each interval and To2 taken as the time for 50% of these cells to become labelled. For recently isolated cells these autoradiographs were also used to determine the proportion of cells in S (DNA synthesis). The labelled fraction of at least 1 000 cells was determined, and the graph of labelled fraction against time was extrapolated to the time the label was added, to give the fraction of cells in S. For established cell lines autoradiographs were also prepared of cells labelled with 5 pCi/ml 3H-thymidine for 30 min at intervals throughout the experimental period and the labelled fraction determined as before. The total cell cycle time (Tc) was measured as described [9]. The non-viable fraction of the population was taken as the constant fraction of the population which did not become labelled after increasing exposure to 3H-thymidine. For established cell lines, Tc, and the fraction of cells in S were corrected for this fraction [lo], but this was not possible for recently isolated cells, and in these cases the values used were uncorrected.

Results TM was recorded for kidney cells after 19, 28 and 69 subcultures. Early prophase was 5 min in duration, but this was a slight underestimate as cells were not always detected right at the beginning of this phase. Late telophase was not timed in all cells but a mean value of 25 min was obtained. After 19 and 28 subcultures the values obtained were in

Preliminary notes Table 2. Population doubling time, labelled fraction and duration of the cell cycle phases of recently isolated Xenopus laevis kidney cells No. of Population subdoubling cultures time (hours)

Duration of phase (hours) ITbelled cells

Cl

28

24.0

15.0

8.0

4.5

30 45

30.0 20.0

14.0 28.4

9.5 11.1

6.0 5.0

37 37

19.0 17.0

22.0 24.0

8.5 6.0 8.0 4.5

44 22 19

24.0 21.5 29.5

25.1 11.3 8.0

11.8 6.6 5.2 5.0 6.0 4.5

27

17.6

15.2

s

G2

0

1

2 3

435

lines. TG2 was about 2 h greater in the tetraploid cells. Cell cycle phase durations of recently isolated kidney cells are shown in table 2 with the population doubling times and the labelled fractions. T,, was approximately constant over these first five subcultures, between 4 and 6 h with the mean value 5.3 h. T, values from 4.5 to 11.8 h were found, and the mean value of this phase was 8.2 h. Gl, always the largest part of the cell cycle, also varied considerably in duration, from 8.0 to 28.4 h. The mean value of the phase was 18 h. Determinations made on recently isolated lung and heart muscle cells gave very similar results.

5 5.4

5.9

The duration of mitosis was assumed to be 0.5 h in all cases.

close agreement. From late prophase to telophase took approx. 30 min; the duration of late prophase itself was 7 min, metaphase 14 min, anaphase and telophase 4 min each. After 69 subcultures the duration of metaphase was reduced by half but the rest of mitosis remained constant. In slower growing cell lines the proportion of mitotic cells was too low to measure TM directly. It was therefore assumed that these values apply to all Xenopus laevis cells in culture, in agreement with approximate values calculated from the mitotic index and T,. The durations of the cell cycle phases of the two established cell lines analysed are shown in table 1 with the corrected and uncorrected parameters on which they are based. For each cell line the values are in good agreement. The duration of DNA synthesis (T,) was greater in the tetraploid cells (EAX 11/75) than in the EAX 09/75 cells (8.9 and 8.2 h as opposed to 6.3 and 6.2 h) while T,, was similar in both cell

Discussion The values obtained for TM in the three cell lines studied indicate that the duration of this phase in Xenopus Zaevis cells in vitro is similar to that observed in mammalian cells in vitro. It is interesting that the cell line maintained longest in culture showed a reduction of the duration of mitosis by halving the duration of metaphase only, suggesting that the other phases of mitosis are recorded as their minimum possible values. Comparing the cell cycle values obtained for tetraploid and aneuploid EAX 09/75 cells, it is clear that doubling the amount of chromatin increased TS by less than half (assuming that the number of chromosomes is an accurate measure of the amount of chromatin to be synthesised). TC was therefore similar in both established cell lines. TC of recently isolated cells was greater than that of established cell lines mainly because T,, was increased. Values of both TG1 and TS increased following the first two subcultures and then decreased as the population became better adapted to culture conditions. After five subcultures, T, was already Exptl Cell Res 87 (1974)

436

Preliminary notes

comparable with that of the established cells analysed. The values of TS determined here are considerably shorter than determined by Malamud [3] for Rana pipiens cells in monolayer culture (22.3 h). It was suggested that this time could be related to the amount of DNA in the Rana pipiens cells. As doubling the number of chromosomes in Xenopus laevis cells does not result in a comparable increase in T,, however, it is possible that there are other reasons for the long TS observed by this author. From the results presented here, it is clear that Xenopus laevis cells in vitro are capable of as rapid DNA synthesis at 25°C as many mammalian cells in vitro at 37°C; similar amounts of DNA are being synthesised in both cases. The other cell cycle phases are also comparable with mammalian cells. If the mammalian relationship between in vivo and in vitro values discussed by Mitchison [l] applies, then the Xenopus laevis cell cycle in vivo may also be similar to mammalian systems. I am grateful to Dr M. Balls for much heluful discussion Gf this work, which was supported-by a grant from the Science Research Council of the United Kingdom.

References 1. Mitchison, J M, The biology of the cell cycle. Cambridge University Press, Cambridge (1971). 2. Flickinger, R A, Freedman, M L & Stambrook, P J, Devel biol 16 (1967) 457. 3. Malamud, D, Exptl cell res 45 (1967) 277. 4. Reddan, J R & Rothstein, H, J cell physiol 67 (1966) 307. 5. Grillo. R S. Oncology 25 (1971) 347. 6. Zalik,‘S E & Yamada, T, J exptl zoo1 165 (1967) 385. 7. Chibon, P, Compt rend hebd seances acad sci 8. 9.

10. 11.

s&r D sci nat 267 (1968) 203. Rafferty, K A, Biology of amphibian tumors (ed M Mizell) p. 52. Springer-Verlag, New York (1969). Godsell, P M. Submitted for publication. Watanabe, I & Okada, S, J cell bio132 (1967) 309. Okada, S, J cell biol 34 (1967) 915.

Received May 5, 1974 Exptl

Cell Res 87 (1974)

Cholera enterotoxin stimulation cultured adrenal tumor cells CATHERINE

of CAMP in

N. KWAN and R. M. WISHNOW,’

Departments of Medicine and Microbiology, University of California, Irvine, and Veterans Administration Hospital, Long Beach, Calif. 90801, USA Summary. Cholera enterotoxin (CT) increased the concentration of adenosine 3’-5’-cyclic monophosphate (CAMP) in monolayer cultures of adrenal tumor cells after a 60 min lag phase in contrast to the rapid effect of adrenocorticotropin (ACTH). The change in intracellular CAMP was accompanied by the release of steroids into the culture medium and a reversible alteration of monolayer morphology.

Cholera enterotoxin (CT) increasesadenosine 3’-5’-cyclic monophosphate (CAMP) in gut mucosa leading to changes in ion transport and fluid accumulation within the intestinal lumen [14]. Recently, several laboratories have shown that cholera enterotoxin stimulates steroidogenesisand alters the morphology of cultured adrenal tumor cells [5-71. The present communication demonstrates that CT increases CAMP after a 60 min lag phase, leading to increased steroidogenesis in cultured adrenal cells. Materials and Methods CeN cultures. Y-l cells, cloned from a mouse adrenal cortex tumor, were obtained from the American Type Culture Collection, Rockville, Md. The cells were maintained in monolayer culture in Eagle’s minimum essential medium (MEM) with Earle’s salts, supplemented with 12.5 % horse serum, 2.5 % fetal calf serum. and 1.0 mM L-alutamine without antibiotics (GIBdo, Grand 1sland;N.Y.). Cells were grown in 60 x 15 mm Petri dishes (Falcon Plastics) at 37°C in a humidified atmosphere of 5 % CO, in air. Time course of ACTH and CT induction of steroidogenesis and CAMP determinations. Growing cultures

(approx. lo6 cells/plate) were washed twice with phosphate-buffered saline. Then Eagle’s MEM without serum containing cholera enterotoxin (50 rig/ml) or adrenocorticotropin (ACTH) (10 mu/ml) was added for 5, 10, 15, 30, 40, 60, 90, 120, or 205 min of incubation. The medium was removed and assayed for 1 Please address requests for reprints to: Dr Rodney M. Wishnow, Medical Research Programs (151), Veterans Administration Hospital, Long Beach, Calif. 90801, USA.