Selection of apoptosis resistant mutant cell lines

Cells to be selected were plated in 96 well, flat bottom ed, plates at 10^ cells per well, m aking a total of 1 x 10^ cells per plate. Each in d ep en d en t pool w as p lated in one 96 well plate per m utagenesis treatm ent. The selection stim ulus was applied and the cells were cultured for 14 days to allow resistant cells to grow out. Successive ro u n d s of m utagenesis and selection w ere carried o u t until three groups of six plates, (one plate per in d ep en d en t pool) w ere generated. After three rounds of m utagenesis and selection, 3 x 10^ cells per pool and a total of 18 x 10^ cells had been m utagenised and selected.

In all experim ents unm utagenised cells w ere selected in parallel for tw o reasons. Firstly, to ensure th at the stim ulation w as effective at inducing ap o p to sis and prev en tin g g ro w th in any w ells an d secondly, to gain an estim ation of the spontaneous rate of m utation in T3.2 cells.

4.3.1 Selection w ith one stim ulus

In o rd er to select m u ta n t cell lines resistan t to the in d u c tio n of ap o p to sis, m u tag en ised cells in 96 well plates w ere selected u sin g the concentration of stim ulus that had been found to effectively induce apoptosis in T3.2 cells after 24 hours (see chapter 3). Typically, the stim ulus w as added to the m edia and the cells cultured for 14 days. H ow ever, in the case of H ydrocortisone and T hapsigargin previous studies h ad show n th at by 16 h o u rs of stim u latio n 100% of the cells h a d en tered ap o p to sis (d ata n o t show n). Therefore, w hen m utagenised T3.2 cells w ere selected w ith these stim uli the wells w ere treated for 16 hours, the stim ulus rem oved an d the

cells then cultured for 14 days. Furtherm ore, this protocol w as sufficient to kill all the control unm utagenised cells. This w as done to m inim ise n o n ­ specific killing of potential m utant cells by the toxicity of H ydrocortisone and T hapsigargin.

4.3.2 Selection w ith tw o stim uli sim ultaneously

The fact th at no m u ta n t cell line g en erated w as re sista n t to an y stim ulus o th er than the one w ith w hich it was selected (figures 4.4, 4.7, 4.10 an d 4.13), indicated th at m utagenesis events w ere occurring u p stream of any

com m on signalling pathw ay. In o rd er to in v estig ate the p o ssib ility of

d e riv in g m u ta n ts in a com m on p ath w ay , m u ta g en ise d T3.2 cells w ere selected w ith tw o different stim uli sim ultaneously. A lth o u g h a ran g e of com binations of apoptotic stim uli w as tested, in all cases no cells grew o u t after 14 day s (data n o t show n). This in d ic ate d th at the p ro to co l w as inappropriate for the derivation of m utants in a com m on pathw ay or th at n o t enough cells w ere screened to find those w ith double m utations.

4.4 G eneration of m utant cell lines resistant to the induction of apoptosis

A sum m ary of the num bers of positive wells w hich were obtained after m utagenesis an d selection w ith each of the individual stim uli is p resen ted in table 4.1. For each of the selection stim uli, a m axim um of 12 in d ep e n d en t w ells w ere picked and grow n up as cell lines for further analysis.

lonom ycin resistan t m utants w ere gen erated from six in d e p e n d e n t groups of cells selected follow ing each of three rou n d s m utagenesis. A fter ro u n d 1 only tw o m utants w ere generated from pool 6. The frequency of m u ta tio n increased after ro u n d 2 treatm en t b u t no fu rth er increase w as observed after round 3. At all times there w ere no positive wells in the plate

co n tain in g u n m u ta g en ise d T3.2 cells in d icatin g th at the tre a tm e n t w as effective at in ducing apoptosis and th at the spontaneous level of m u tatio n w as below background. Wells w ere chosen from different pools including one w ell from ro u n d 1. A total of tw elve wells w ere picked an d tested for resistance to lonom ycin-induced apoptosis (section 4.5.1). The resulting cell lines w ere n o t subcloned at this stage.

A t the first attem pt to generate Con-A resistant m utants, no positive wells w ere obtained following any of the m utagenesis rounds indicating th at the m utation rate w as very low. Therefore the experim ent w as repeated and the cells w ere not selected until they had had all three cycles of m utagenesis treatm ent. After 3 rounds of m utagenesis, there w ere a significant num ber of w ells w ith Con-A resistant cells growing. Pool 2 h ad the m ost positive wells a n d th erefo re it w as possible th at these co n tain ed sib lin g m u tatio n s. Therefore all the w ells from pool 6 and pool 1 w ere picked for fu rth er analysis together w ith four from pool 2. The cell lines w ere th en tested for resistance to Con-A-induced apoptosis (section 4.5.2).

In the first experim ent w here T3.2 cells w ere m utagenised and selected w ith H ydrocortisone, the cells failed to recover after round 2 treatm ent and so no selection could be applied. H ow ever, one well from ro u n d 1 w as positive an d w as isolated. Repetition of the experim ent gave no positive wells after ro u n d s 1 an d 2 w ith two wells positive after ro u n d three w hich w ere also

isolated. The cell lines w ere then tested for resistance to H ydrocortisone-

induced apoptosis (section 4.5.3).

It can be seen in table 4.1 th at after three ro u n d s of selection w ith T hapsigargin only pools 1 and 5 show ed any signs of growth. N o wells were positive after rounds 1 and 2. As there were less th an 12 wells in total w ith signs of cell grow th all 7 w ells w ere picked and assessed for resistance to T hapsigargin-induced apoptosis (section 4.5.4).

In order to have a clear record of the source of each m u tan t cell line, a sim ple nom enclature system for m utants w as designed. Each m u ta n t w as assigned a letter denoting the stim ulus w ith w hich they h ad been selected (I - lonomycin, C - Con-A, H - Hydrocortisone and T - Thapsigargin). Then each m u tan t w as given the ro u n d num ber, the pool from w hich it w as derived and the well location. For exam ple m utant I16A10 is an lonom ycin resistant m u tan t derived after round 1, from pool 6, well AlO. Table 4.2 show s a list of all the m u tan t cell lines isolated.

4.5 Cell death in apoptosis resistant m utant cell lines

To characterise the n atu re of the m u tatio n s d eriv ed , cell d e ath in m u tan t cell lines was analysed in a num ber of ways. Firstly, the phenotype of the cell lines w ith respect to CD3, CD4, CDS an d HSA w as exam ined by staining and flow cytom etry to investigate w hether any change in cell surface m arkers had occurred. The induction of apoptosis in thym ocytes prim arily occurs in double positive, CD4, CDS cells (chapter 1). If the m u tag en had caused some form of cell m aturation, the cells could have lost sensitivity to the in d u ctio n of apoptosis w ith o u t m u tatio n of the ap o p to tic p ath w ay . Therefore it was necessary to determ ine the phenotype of all m u tan t cell lines to exclude this possibility.

Secondly, there was a possibility that the karyotype of m u tan t cell lines h a d changed as a resu lt of m utagenesis. G ain or loss of a chrom osom e containing genes involved in apoptosis had the potential to greatly affect the induction of apoptosis by various stimuli. A lthough chrom osom e loss w as unlikely, given the w ay that the m utagen operates, each m utant cell line w as characterised w ith respect to its chromosome content using the sam e protocol as used for T3.2 cells (section 3.2).

T hirdly, in o rd er to confirm th a t the m u ta n ts w ere re s ista n t to induction of apoptosis by the selecting stim ulus they w ere exam ined for cell grow th (3RT incorporation) and DNA fragm entation (7AAD staining) after 24 hours of stim ulation. Both of these assays w ere used to ensure th at apoptosis w as occurring and to get an accurate m easure of the percentage of apoptotic cells after treatm ent.

Fourthly, in order to investigate the n atu re of m utations, m u ta n t cell lines w ere tested for resistance to the induction of apoptosis b y the other stim uli w ith w hich T3.2 was characterised. The cell lines w ere tested by analysing DNA fragm entation after treatm ent for 24 hours and th en if any

resistance w as observed the cell line w as tested w ith ^h T in co rp o ratio n to

gain a fuller picture.

4.5.1 Induction of apoptosis in lonom ycin resistant m utant cell lin es

In order to stu d y the phenotype of the four m u tan t cell lines w hich w ere fully resistant to the induction of apoptosis by lonom ycin (see below ), cell lines were stained for CD3, CD4, CD8 and HSA. I16A10,123D4 an d I34D9 displayed indistinguishable staining w hen com pared to T3.2 cells and so only

I16A10 an d I32F7 are show n in figure 4.2. A lthough I32F7 h a d altered

expression of the co-receptors CD4 and CD8, unaltered expression of HSA and CD3 indicated th at it still ap p eared to have an im m atu re p h en o ty p e. In addition, analysis of the m utant karyotypes revealed that no gross karyotype changes had occurred during m utagenesis (data not shown).

The twelve isolated (section 4.4) cell lines w ere tested for resistance to lonom ycin-induced apoptosis by ^HT incorporation to confirm th at they were m utants. Only four cell lines (I16A10, I23D4, I32F7 an d I34D9) from the

tw elve picked w ere found to be b o th fully resistant to lonom ycin-induced apoptosis an d to be derived from independent pools (data n o t shown). The rem a in in g e ig h t w ere either p a rtially re sista n t or n o t re sista n t w h e n com pared to T3.2 cells.

M utants w ere re-tested for the in duction of apoptosis b y m easuring 3RT incorporation in parallel w ith assaying DNA fragm entation by 7AAD

staining. Figure 4.3a show s th at there is no g ro w th a rre st in d u ced by

lonom ycin in I16A10, I23D4,132F7 and I34D9 m utant cell lines after 24 hours w h en com pared to T3.2 cells. Figure 4.3b confirms th at in addition to grow th a rre s t, lo n o m y c in is in cap ab le of in d u c in g an y in cre ase in D N A fragm entation after 24 hours in these cell lines. This finding confirm s th at the cell lines are com pletely resistan t to the in d u ctio n of ap o p to sis by lonomycin. It w as possible that the m utants were m erely delayed in the onset of apoptosis after treatm ent. H ow ever it w as found th at treatm ent for u p to 72 hours gave the same result (data not shown).

In o rd e r to characterise the n atu re of the m u ta tio n s w h ich h a d occurred, lonom ycin resistant cell lines w ere taken and treated for 24 hours w ith 10 p g /m l Con-A, 1 pM H ydrocortisone and 50 nM T hapsigargin. The cells w ere th en perm eabilised, stained w ith 7AAD and analysed. Figure 4.4 show s th at all except one of the lonom ycin resistant m u tan t cell lines show no increased resistance to apoptosis induced by any other stim ulus tested. The bottom category of each graph denotes the percentage of increased DNA frag m en tatio n induced by each stim ulus in p aren t T3.2 cells. In all cases, except one (I32F7, discussed below) the DNA fragm entation induced is exactly com parable to T3.2 cells.

Figure 4.4 indicated that there was a significant reduction of in duced D N A frag m e n ta tio n in I32F7 cells trea te d w ith 1 |iM H y d ro c o rtis o n e com pared to T3.2 cells. This is show n in m ore detail in figure 4.5a w hich show s th at there is a significant percentage (37%) of cells rem aining in cell

cycle after I32F7 cells have been treated (right) w ith 1 \lM H ydrocortisone. It appears th at the cells, analysed by 7AAD, w hich rem ained in gate 1 w ere not apoptotic as there ap p eared to be cells in S phase. H ow ever, to fu rth er characterise this phenom enon, the effect of 1 pM H ydrocortisone o n grow th arrest in I32F7 cells w as investigated. Figure 4.5b show s that, alth o u g h a significant drop in ^HT incorporation was observed, the counts did not drop below background as w as observed in T3.2 cells (figure 3.8a). This indicated th at the cells in gate 1 after treatm ent, figure 4.5a, cells w ere in cycle and that I32F7 had partial resistance to apoptosis induced by H ydrocortisone. W hether this extra resistance w as due to m ultiple m utations or one m utation affecting both pathw ays was unclear at this stage.

In sum m ary, lonom ycin resistant cell lines behaved the sam e as T3.2 cells to all stim uli except that they are completely resistant to the induction of apoptosis by lonomycin. The exception is I32F7 w hich has altered expression of CD4 an d CDS and is also p artially resistant to apo p to sis in d u ce d by H ydrocortisone.

4.5.2 Induction of apoptosis in the Con-A resistant m u tan t cell lin e C36H12

G row th arrest induced by Con-A w as exam ined by m easu rin g % T \

incorporation in all tw elve isolated cell lines. Eleven cell lines show ed either partial or complete grow th arrest w ith only one line, C36H12, displaying no grow th arrest (data not shown). C36H12 was analysed in m ore detail and also tested for DNA fragm entation by 7AAD staining. Figure 4.6a show s th at w hile T3.2 cells exhibit complete grow th arrest, C36H12 does not. This is confirm ed by the lack of DNA fragm entation in these cells after Con-A treatm ent (figure 4.6b).

In o rd er to characterise fu rth er the lesion o bserved in the C36H12 m utant, it was tested for DNA fragm entation by 7AAD staining after 24 hours

treatm en t w ith lonom ycin. H ydrocortisone an d T hapsigargin. Figure 4.7 show s th a t C36H12 cells enter apoptosis in a sim ilar fashion to T3.2 cells w h en treated w ith stim uli other than Con-A.

Analysis of surface m arkers on the Con-A m u tan t C36H12 show ed that it w as u n c h an g e d from T3.2 w ith respect to CD3, CD4, CD8 an d HSA expression and furtherm ore the karyotype of this cell line w as the sam e (data not shown).

In sum m ary, C36H12 is unchanged from T3.2 cells in its response w ith respect to all stimuli, m arker expression and karyotype except for the fact they no longer en ter apoptosis in response to Con-A. The exact n a tu re of the m utation(s) rem ain unclear b u t it appears th at the m utation(s) are u pstream

of any conversion point into a comm on pathw ay. A d d itio n a lly , C 36H 12

cells aggregated into clum ps w hen treated w ith Con-A in a m anner sim ilar to