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week, the cells are very dense and have aggregated. It should be borne in mind that these cells have been cultured in the standard imaginai disc cell line culture medium which contains 1 ng/ml (2 x 10"^ M) 20-HE. Fig. 4.8.2 shows a culture that was grown with 2 ng/ml (4 x 10"^ M) 20-HE, the cells appear

less dense than in the control (Fig. 4.8.1) and although they have aggregated do not form the large conglomerated aggregates seen in Fig. 4.8.1. A culture grown with 4 ng/ml (8 x 10“® M) 20-HE is shown in Fig. 4.8.3. The cells are much less dense but aggregates of cells have formed. Fig. 4.8.4 shows a culture that has been incubated with 20-HE at a level of 6 ng/ml (1.2 x 10”® M), again the cells appear to be less dense and the aggregates are bigger and more definite with cell processes radiating outwards. At 8 ng/ml (1.6 x 10“® M) in Fig. 4.8.5 this process can be seen to have gone further.

There are now large areas of bare tissue culture plastic, the aggregates are more defined and the cell processes extending from these aggregates are longer. Fig. 4.8.6 shows a culture incubated with 10 ng/ml (2 x 10“® M) 20-HE for a week.

Therefore it seems that even at much lower

concentrations of 20-HE down to 2 ng/ml the cells respond. It seems that the reduction in cell division rate is the most sensitive part of the cells response to 20-HE occurring even at 2 ng/ml. Aggregation also seems to be one of more sensitive rësÿdnses of the cells, being commonplace at 4 ng/ml. Cell processes radiating from aggregates seem to be more prevalent

at higher levels of 20-HE although cell elongation seems to occur at lower 20-HE titres (4 ng/ml).

Therefore it seems that at lower levels of 20-HE the response of the cells is progressively reduced. Again the cessation or reduction in cell division is characteristic of both low levels of 20-HE and also in cultures briefly exposed to an optimal concentration. This indicates that this

cessation of cell division appears to be a very early and primary action of 20-HE, followed later by cell elongation, aggregation and process extension.

This response to 20-HE is apparent in both wing and leg imaginai disc cell lines and also in nearly all of the clones so far isolated from these primary lines. However there are a few exceptions to this. For instance the leg lines so far tested (3) whilst showing all the features that have been mentioned previously do appear to be somewhat more sensitive to 20-HE that wing lines. This can be seen in Fig. 4.9, which shows the effect of administering a 5 ng/ml (1 x 10”® M)

concentration of 20-HE to two cloned cell lines, one wing and one leg. Figs. 4.9.1 and 4.9.2 show the control and

experimental respectively for the wing line C1.8+. This produces a response which has been seen before in the low titre experiments, the cells are much less dense, and there are several aggregates. Although individually some of the

cells have elongated there does not appear to be large numbers of processes radiating from aggregates as seen for the 10

ng/ml treatment.

In contrast Figs. 4.9.3 and 4.9.4 show the control and experimental for the cloned leg line Ll Astrew (LIA). The

difference between the control and 5 ng/ml treatment is more stark than for the cloned wing line C1.8+, 5 ng/ml induces a similar response in the line as 10 ng/ml of 20-HE in the Cl.8+ cell line. The cells have aggregated in places, there are some areas of bare plastic and there are numbers of cell processes extending across the plastic. Note the flattened out cells

(arrowed), these are often seen in 20-HE treated cultures.

One cell line produces a response which is significantly different from that which has been described above. The cloned wing cell line 09 shows an apparent insensitivity to the

effects of 20-HE. Fig. 4.10 shows a culture of 09 treated with 100 ng/ml (2 x 10”? M) of 20-HE for a week. At this level

other cloned and uncloned cell lines would be very severely affected, most of the cells would have died and there would not be the extensive aggregation and cell process extension that is seen at lower 20-HE titres. However this cell line seems to be relatively unaffected by this extremely high level of 20-HE. There does appear to be some 20-HE this cell line, albeit very much reduced, the cells appear somewhat less dense than in the control and there appears to have been some

aggregation.

Apart from this particular cell line all the lines so far tested have proved positive for the action of 20-HE. Even this cell line C9 does show some effect and is not completely insensitive to 20-HE. Thus besides the above mentioned

differences the effect of 20-HE on all the cell lines tested was quite similar. Occasionally in culture patches of tanned cuticle-like material would be produced. This phenomenon was

most common in the cloned leg cell line LIA but was also

apparent in other cell lines (Fig 4.11). |

When conducting these experiments on the effects of ecdysteroids, the cultures were carried on beyond 7 days and any different effects observed. The 20-HE response did not change appreciably beyond this period and therefore 7 days was taken as a convenient incubation period for 20-HE to take

effect. However occasionally after a while some cells began to divide again in the 20-HE treated cultures. Fig. 4.12 shows an aggregate where cells have begun to divide again. These cells were still growing in a medium containing 20-HE so it appeared that they had overcome the growth inhibiting effects of 20-HE. When a small group of these cells were observed, the cultures were allowed to continue growth and the medium changed to one without any 20-HE. This allowed any cells that had survived 20-HE treatment such as the small group in Fig. 4.12 to grow up and form a culture. This was repeated with alternating periods of no 20-HE and medium with 20-HE for a period of 2-3 months. During this period the 20-HE concentration was

gradually increased during periods of 20-HE exposure.

This regime specificially selected for cells that were resistant to the effects of 20-HE based on their ability to

continue dividing in medium containing 20-HE and without .■ showing any morphological alterations. Eventually cells could ! be grown in medium containing 1*5 ug/ml of 20-HE, with no

effect. Thus cells had been selected for 20-HE resistance. The ; 9 effects of such selection can be seen in Fig. 4.13.(1-4). This j shows the wing line Cl.8+ subjected to increasing levels of

20-HE (10-150 ng/ml (2 x 10”® M - 3 x 10”? M)) after 7 days. 1

This line was also used to select cells that were resistant to 20-HE and a line was grown up termed C1.8R which was resistant to 20-HE. In Fig. 4.13.(5-8) increasing levels of 20-HE to 150 ng/ml (3 x 10”? M) can be seen to have no effect on the cells whereas in Fig. 4.13.(1-4) increasing levels of 20-HE can be seen to have a dramatic effect on the parent line C1.8+.

Thus by the use of an appropriate selective pressure, in this case increasing 20-HE levels, cells have been selected for resistance providing a useful control for a number of 20- HE experiments, especially those associated with biochemical aspects of 20-HE action.