Obras por administración directa:

Under this heading two assays were employed: pigeon red cell (Section 3.1.2) and assessment of cell number using coulter counter (Section 3.1.3).

Prior to setting up the pigeon red cell (DiSC) assay the methodology was evaluated by determining the ratio of live and dead cells in a mixed population (Section 3.1.1). The responses of the following leukaemic cell lines to Ara-C, mitoxantrone either singly or in combination were determined using both the DiSC assay and assessment of cell number using coulter counter: SA2, high cell dose (HD) transplant of SA7 (SA7HD) and SA8 high cell dose passage (SA8HD).

3.1.1 Evaluation of Pigeon Red Cell (DiSC) Method: Leukaemic bone marrow single cell suspension was prepared from femora of mice bearing the SA7HD leukaemia as described under section 2.1.3. Some of the cells were killed by heating at 60 °C for one hour and mixed with live cells to give 0, 25,50, 60, 80 and 100% live cells. Viability was determined after staining with fastgreen-nigrosin and counterstaining with Jenner- Giemsa.

Results: There was a good correlation between expected and observed values (Figure 1) and this indicated that the procedure does preserve and differentiate live and dead cells in a mixed population. Live cells assumed normal Jenner-Giemsa counterstain (pink) while dead cells were stained dark-green by fastgreen-nigrosin(Plate 1).

3.1.2 Pigeon Red Cell (DiSC) Method: A single cell suspension was prepared from the following leukaemic cell lines (SA2, SA7HD, SA8HD) and plated at 7 x 1 0^ cells per well in a microtitre plate. W EHi conditioned medium was added (except cells from SA2 cell line) to give a final concentration of 10% per well. lOOpl each of mitoxantrone, Ara-C or combinations of the two was added to give final concentrations in the range 0.12-120ng/ml. In combination experiments, the concentration of one drug was varied (0.1 2-1 2 0ng /m l) while maintaining the final concentration of the second drug at 1.2ng/m l. After four days incubation, six wells were pooled and the cells were stained with fastgreen-nigrosin to which permanently fixed pigeon red cells were added. They were then cytocentrifuged onto ethanol-washed slides. After Jenner-Giemsa counter staining, the ratio of live cells over simultaneously counted pigeon red cells was determined (blindly) for each slide and the ratio in drug-treated samples expressed as a percentage of that in control. This expression called tumour cell viability (TCV) was plotted against drug concentrations to yield dose-response curves.

3.1.3 Assessment of Cell Number: Experiments were set up as described under section 3.1.2. On day 4 following drug incubations, six wells were pooled (each) from drug treated and control samples and counted using a coulter particle counter (see Section 2.2.3). Dose-response curves were obtained by plotting the ratio of counts in drug treated samples as a percentage of that in control as function of drug concentrations.

Results: The responses of the leukaemic cell line SA2 to Ara-C alone and in combination with mitoxantrone (1.2ng /m l determined using both pigeon red cell (DISC) and coulter (CC) methods are shown in Figure 2. According to both methods, the cell line was not sensitive to Ara-C w ithin the concentration range 0.1 2-1 2n g /m l as only slight

increase in cytotoxicity was observed with increasing dose within that range. However, a marked cytotoxic effect was seen with 120ng/ml in the D isc assay. There was a close agreement between the two methods although the electronic particle count method seemed to underestimate

1001 90 - 80 - 70 - 0 Expt 1 ♦ Expt 2 ■ Expt 3

m

0

_{Live cells expected(%)}

Fig.l : Percentage of live cells observed after staining known proportions of live and dead cells with fast green.

120 -I _{arac+Mitox( 1.2ng/m1)(DiSC)}arac (DiSc) aracCC.C) arac-i-initox(1.2ng/m1)CC.C} 100 r-'TTi rm mitox 1.2ng/m1 I I 1 I i i | I- .1 1 10 too 1000

Concentration of arac(ng/m1)

Fig.2: The response of SA2 leukaemic cell line to arac alone or in combination with mitoxantrone assesed using the DISC assag

and coulter count method.

cytotoxicity produced using the highest concentration employed (120ng/m l). W hilst a slight additive effect was observed when mitoxantrone (l,2ng/m l) was added to Ara-C (in the concentration range 1.2-12ng/ml) in the DiSC assay, no such effect was observed using the coulter method. In the latter case where mitoxantrone was added to Ara- C, there was no difference from the response with Ara-C alone. Figure 3 shows the effect of mitoxantrone alone or in combination with Ara-C (1.2ng/m l) on the same (SA2) leukaemic cell line using both methods. Again there was close agreement between the dose response curves obtained using the two methods. As was observed with Ara-C, mitoxantrone within the concentration range 0.1 2-1 2ng/m l had virtually no effect on cytotoxicity. However, increased cytotoxicity was observed when higher concentration of mitoxantrone was used (1 2 0ng /m l). Mitoxantrone alone was no better than Ara-C alone in the extent of cell kill produced. Addition of Ara-C to mitoxantrone resulted in either no

effect (cell count) or antagonism at low concentrations of mitoxantrone (DISC).

The responses of the SA7 cell line to Ara-C alone and in combination with mitoxantrone are shown in Figure 4. This cell line seemed slightly more sensitive to Ara-C alone as compared to the SA2 cell line using the cell number assessment method. There was a close agreement between the two methods in determining the response of the cell line to Ara-C alone. Addition of mitoxantrone (1.2ng/m l) to Ara-C resulted in either no increased effect (DISC) or an additive effect (CC). This cell line seemed more sensitive than SA2 to mitoxantrone alone and in combination with Ara-C (Figure 5) as assessed by both DISC and cell number assessment methods. Addition of Ara-C to mitoxantrone resulted in antagonism at the lowest mitoxantrone concentration (0.12ng/ml)(DiSC) or slight additive effect (CC). Figure 6 shows the dose- response curve of the SA8 cell line to Ara-C alone and in combination w ith mitoxantrone. A t low to intermediate concentrations (0.12- 12ng/m l), Ara-C had little effect on cytotoxicity as assessed by both methods. When the dose of Ara-C was increased to 120ng/ml there was about an 80% increase in cytotoxicity relative to control. Both methods gave almost identical dose-response curves. Addition of mitoxantrone to Ara-C, resulted in additive cytotoxic effects as determined using both methods. The SA8 cell line is as sensitive as the SA7 cell line to mitoxantrone (CC) (Figure 7). Unlike what was observed with Ara-C, increasing the concentration of mitoxantrone w ithin the range 0.1 2- 12ng/m l was associated with increased cytotoxicity.

S % be VW 100 - arac d -2ng/m | ) 60 40 9O U 20 L.e S 0 - M itox(D iSC) » M itox(C .C ) " •••■■♦•... M ito x + A ra c d .2ng/m O (DiSC) ❖--- MItoxHhAracd .2ng/in1)(C .C ) 1---1----i r I I M |--- 1---1 I I I Im " ■ I I I r T 'T I H ' I 1 !■ I t I T U 1 1 10 100 1000

Concentration of mitox(ng/m1)

Fig.3: The responses of SA2 leukaemic cell line to mitox alone or In combination vith

arac assessed using the DISC assag and the coulter count method

120 -1

100

o 80

(J 20

arac(DiSC)

arac* mi tox( 1.2ng/m l)( DISC) arac(C.C)

—^— arac+mitoxd .2ng/ml)(C.C> t mitox

d 2ng/m l)

t— I I I I I I— r " T " r i m | i - i —» "i ï i u | r i— i i t n 'T |

.1 1 10 100 1000