Introduction

D ouble positive thym ocytes are very sensitive to the in d u ctio n of apoptosis by a variety of stim uli (Migita et al., 1994) an d therefore a good m odel in w hich to study the biochemical and genetic basis of apoptosis. In o rd er to stu d y the induction of apoptosis and to have a system am enable to genetic m an ip u latio n , I decided to find a cell line w h ich m irro re d the apoptotic behaviour of isolated thymocytes and w hich self-perpetuated to use as a m odel w hich w ould lend itself to genetic m anipulation.

Mice transgenic for c-myc u n d er the control of Thy-1 prom oter and

control elem ents get thym ic tum ours w hich can be grow n o u t in vitro. The

over expression of c-myc, in addition to its ability to cause transform ation in

m an y cell types, has been show n to ren d e r cells m ore sensitive to the in d u ctio n of apoptosis by m ethods such as serum starv atio n (Evan et al., 1992). Therefore it was considered possible that a cell line driven by the over

expression of c-myc w ould be very sensitive to the induction of apoptosis and

ideal for these studies. A num ber of thymocyte tum our cell lines from these mice w ere screened and the cell line T3.2 was found to be the m ost sim ilar in ap o p to tic b eh av io u r to iso lated thym ocytes an d w as chosen to be the experim ental system.

The first step w as to characterise the cell line in detail w ith respect to the occurrence of apoptosis. Multiple assays were used in order to ensure that the cell d eath w hich w as observed w as b y apoptosis, an d to gain a fuller picture of the occurrence of different features of apoptosis su ch as gro w th arrest and DNA fragm entation. Apoptosis has been show n to be induced by a variety of stim uli w hich in tu rn appear to utilise a range of different signal tran sd u ctio n p ath w ay s (McConkey et al., 1994). In o rd er to com pare the behaviour of T3.2 to isolated thymocytes as fully as possible, and to stu d y the

different signal transduction pathw ays, several stim uli w ere u sed to induce apoptosis in T3.2 cells. These w ere lonom ycin, PdBu, Con-A, anti-CD 3, H ydrocortisone, Thapsigargin and Staurosporine

T3.2 cells w ere found to undergo apoptosis in response to lonom ycin, Con-A, H ydrocortisone and T hapsigargin and w ere also fo u n d to readily undergo cell death in response to Staurosporine. In contrast, T3.2 cells w ere found not to undergo apoptosis in response to PdBu and plastic cross-linked anti-CD3.

3.2 D erivation and phenotype of T3.2

The cell line T3.2 w as derived by Yujiro Tanaka (M urdjeva et al., 1996) from a cross betw een a T cell receptor transgenic m ouse (F5) an d a Thy-myc

m ouse. Thy-myc mice express c-myc under the control of the Thy-1 prom oter

an d control elem ents. These mice generally develop thym ic tu m o u rs 6-12 w eeks after b irth (Spanopoulou et al., 1989). One such tu m o u r w as teased ap art in tissue culture m edium and then cultured at 37°C until cell lines were established. The cells w ere then sub cloned and the in d iv id u al clonal lines characterised.

The first step to determ ine w hether T3.2 w as a suitable m odel for double positive thym ocytes w as to establish the p h en o ty p e of T3.2 cells. Double positive thymocytes express high levels of HSA, CD4 and CDS and an in term ed iate level of CD3 com pared to m ature T cells (K ruisbeek, 1993; Janew ay and Travers, 1994). T3.2 cells were taken in log phase and analysed for CD3, CD4, CDS and HSA expression by flow cytometry. Figure 3.1 shows the expression of CD3 (a), CD4 (b), CDS (c) and HSA (d) by T3.2 cells. T3.2 cells express CD4, CDS and HSA at a high level, com parable to im m ature double positive thym ocytes, indicating th at T3.2 m ay be derived from the desired thym ocyte sub-population. In ad d itio n CD3 expression on T3.2 cells w as

com pared to th at of BIO thymocytes. T3.2 show ed a bim odal p attern of CD3

staining. The m ajority of cells expressed levels of CD3 com parable to

im m ature thym ocytes, figure 3.le and 3.If, w hile the rem ain d er expressed

less. These figures show th at the low er level of exp ressio n of CD3 on

thym ocytes (im m ature population) is comparable to th at on T3.2 cells. This d u al p o p u latio n w as found to be stable and if T3.2 cells w ere sorted into discrete populations of CD3 expression, the lines retu rn ed to dual expression

w ith in a few m onths. A poptosis w as fo u n d to be id en tical in eith er

population indicating that CD3 expression was incidental in this system (data not shown).

In order to further characterise T3.2 cells and to assess w h eth er any m ore genetic changes had occurred d u rin g transform ation, the n u m b er of chrom osomes per m etaphase nucleus was counted. T3.2 cells w ere taken and

blocked from leaving m etaphase w ith demecolcine for 2 ho u rs to m axim ise

the num ber of cells in mitosis. The cells w ere then sw ollen in a hypotonic solution, fixed an d d ro p p ed onto a slide causing the m etap h ase nuclei to ru p tu re , sp read in g the chrom osom es so they could be counted. Geim sa stain ed slides w ere m o u n ted an d counted at lOOx u n d e r oil im m ersion. Figure 3.2 show s a photograph of a typical field and below a g rap h show ing the percentage of cells containing different num bers of chrom osomes. 75% of T3.2 cells contain a total of 42 chrom osomes w hich is tw o m ore th an norm al m ouse cells ((Robertson, 1987)). The rem ainder h ad a norm al karyotype. It could not be determ ined w hich chrom osomes w ere duplicated and w hether

any o th er anom alies w ere p resen t u sing this assay. A lth o u g h it is n o t

uncom m on for cell lines to contain karyotype irregularities (R udniki and McBurney, 1987), the significance of the tw o extra chrom osom es w ith respect to apoptosis rem ains unclear.