DISCUSIÓN Y RESULTADOS.
4.2.3.4 PRINCIPIO DE FLEXIBILIDAD a Ley 29785 Ley del Derecho a la Consulta.
presence of increasing concentration of recombinant IL2 and
allowed to grow for 20 hours. IjiCi of
thymidine was
added to each culture and cells grown for an additional 4
hours before harvest and counting. Labelled DNA was
harvested onto glass fiber filters and counted in scintillation
fluid.
in cell cycle analysis, is m em brane im perm eant, and only has access to DNA if m em branes are leaky - this can occur either by fixing the cells, or though cell death. O ne characteristic of apoptotic cells is th at they possess intact cell m em b ran es. O nly in later stag es of d e a th do m em b ran e s becom e com prom ised. The second dye used, Hoechst 33342, is a Vital' dye that is taken up preferentially by apoptotic and dead cells. Thus, the blue fluorescence intensity of Hoechst in live cells is less than that of apoptotic cells, and can be distinguished by FACS. Apoptotic cells, therefore, are distinguished from dead cells by virtue of Hoechst versus PI staining, apoptotic cells show ing intense H oechst and low PI staining, dead cells show ing intense H oechst and PI, and live cells show ing low Hoechst and low PI staining(312, 313).
The results of cell cycle analysis on CTLL are show n in figure 6.2 and table 6.1.
Table 6.1: The effect of IL2 upon CTLL cell cycle
IL2, n g /m l % Gi^ % G2/S /M 0 55 38 1 48 49 5 45 54 10 45 55 2 0 43 57
^These num bers are derived from the m arkers as suggested in figure 4.2; M l= % G i,M 2 = G2/S /M .
It is apparent that the dose of IL2 does not appear to have a significant effect u p o n the cell cycle status of CTLL. Thus, w ith d raw al of lym phokine and subsequent decrease in 3H Tdr uptake does not correlate w ith a change in cell cycle state of CTLL. U sing ju st cell cycle analysis, it appears th at w hen
200 4 0 0 6 0 0 1000 200
M ELISSA:DC 19 0 2 0 1 8 \ F 3 2 - H \ F 3 2 - H e i g h t
200 6 0 0 8 0 0 1000 200 4 0 0 6 0 0 10 0 0
MEL 1SSA: D C l9 0 2 0 1 6 \ F 3 2 -H N F 3 2 - H e ig h t
200 6 0 0 8 0 0 1000
F ig u re 6.2: T h e effect of ly m p h o k in e d e p riv a tio n o n cell cycle sta tu s of CTLL. CTLL w ere d e p riv e d of IL2 as p rev io u sly d escribed, a n d g ro w n in the absence o r in creasin g co n cen tratio n of IL2 for 24 hours: A = O ng/m l, B = ln g /m l, C = 5 n g /m l, D = 1 0 n g /m l, an d E = 2 0 n g /m l IL2. Cells w ere p e rm eab ilized a n d stain ed w ith p ro p id iu m io d id e to label DNA. Cell cycle analysis w a s p e rfo rm ed as described in M aterials an d M ethods, v isu alizin g fluoresence u sin g FACStar. The d a ta are d isp lay e d as relative cell n u m b er (y-axis) v e rsu s red (PI) fluoresence in ten sity on th e x-axis. N ecrotic cells w ere excluded b ased on FSC a n d SSC characteristics. T he m ark ers sh o w n on each g rap h c o rresp o n d to M l: a n d M2: G2/S /M . The
lym phokine is w ithdraw n, the cells sim ply arrest in w hatever state of the cell cycle they are in to wait for the addition of m ore IL2.
W hen apoptosis of these cultures w as exam ined, it becam e ap p aren t th at w ith d raw al of lym phokine w as having a dram atic effect u p o n the cultures. Thus, at the same time that cells were analyzed for cell cycle in figure 6.2, a p o rtio n of the culture w as taken and dual-stained w ith H oechst 33342 and propidium iodide, and percent of cells in apoptosis exam ined. The results are illustrated in figure 6.3 and tab le 6.2. The histogram s depicted in fig u re 6.3 show PI staining on the x-axis and H oechst staining on the y-axis. In fig u re 6.3E, two predom inant populations are evident. The cells in region 1 - 'R l' - show low intensity staining for both PI and H oechst, suggestive of live, grow ing cells. The cells in region 3 - R3' - stain brightly w ith PI and also w ith Hoechst, w hich indicates they are predom inantly dead. A th ird region, R2, defines apoptotic cells, which characteristically take up Hoechst quickly(within tw o m in u tes of application), b u t do not exhibit stain in g w ith PI. This population is not seen in 6.3E, but is increasingly obvious as the concentration of IL2 is decreased (figure 6.3E-A and table 6.2). Apoptotic cells are first detected
Table 6.2: Apoptosis in IL2 deprived CTLL cells.
IL2, n g /m l R ia R2 R3 0 1.09 30.85 65.56 1 64.41 7.02 23.38 5 80.44 0.60 13.97 10 84.63 0.32 10.21 2 0 84.66 0 .1 0 6.57
MELISSA:DC1908006 MELISSA:DCl9 8 2007 50% Lofl i è ‘ F L 2 - H x F L 2 - H e i g h t — > M E L IS S A :D C 1902008 50% Lofl T è ° i 'è ' i'Ô2 F L 2 -H N F L 2 -H e i0 h t ---> MEL ISSA :D C 1 9 0 2 0 1 0 lô'^ 50% L o n i è ° i é ‘ i è î F L 2 -H N F L 2 -H e ig h t ---> 50% Lofl 10» i’è î F L 2 - H v F L 2 - H * i 9 h t ---> MEL IS S A I DC1 9 0 2 0 0 9 50% L o a lA o 10» F L 2 - H \ F L 2 - H * i g h t
F ig u re 6.3: A p o p to sis in IL2-fed or IL 2 -d ep riv ed CTLL.
CTLL c u ltu res from figure 6.2 w ere used for ap o p to sis analysis. Cells w ere sim u lta n eo u sly stain ed w ith p ro p id iu m io d id e an d H oechst 33342 for tw o m in u te s an d th en an aly zed w ith a d u a l laser as d escrib ed in M aterials an d M ethods. In each plot, H oechst staining is rep re se n te d logarithm ically on the y-axis w ith p ro p id iu m iodide staining on the x-axis. R elative cell n u m b ers are rep re se n te d by the contours. S u b p o p u la tio n s in each c u ltu re are identified by d en sity of contours. Boxes o u tlin in g su b p o p u la tio n s w e re d ra w n to give a p ercen tag e of the total, as d iscu ssed in the text. C o n cen tratio n of IL2 in A-E are
^N um bers show n are the percentage of each cu ltu re fo u n d in the area represented by the 3 regions in fig u re 6.3. R2 and R3 w ere defined on the O ng/m l culture, while R l was defined on the 2 0 ng/m l culture.
in great num bers in cultures having only I n g /m l IL2(figure 6.3B). This is
rem in iscen t of the half-m axim al dose of IL2 req u ire d for p ro liferatio n , ~2ng/m l(figure 6.1). In cultures grow n in the absence of IL2, as seen in fig u re 6.3A, a great proportion of the cells are dead, but a significant num ber are also in apoptosis, as judged by Hoechst staining seen in the cells in R2. Thus, Tdr uptake in CTLL is more a reflection of viability of CTLL, rather than of cells in S phase, and the major effect of IL2 deprivation on CTLL is cell death, caused by apoptosis.
6.2: Transient expression o f proteins in CTLL.
Prelim inary experim ents suggested that CTLL could readily be transfected, as judged by the expression of the reporter gene CAT from different heterologous p ro m o te rs (data n o t show n). To d eterm in e w h e th er v -H a-ras could be functionally expressed in CTLL, we perform ed a co-transfection experim ent using, as a reporter for Ras activity, AP-1 CAT. As dem onstrated in figure 6.4, AP-1 CAT has very low basal activity, which can be induced by treatm ent of cells w ith phorbol esters. In addition, IL2 w as also ad d ed as a source of stim ulation, and resulted in a 3 fold increase in activity. In cells co-transfected w ith AP-1 CAT and v-ras, a m arked increase in reporter gene activity, in this experim ent nearly ten fold, can be detected. Thus, the v-H a-ras protein can be expressed transiently in CTLL, and is functional.
One concern about studying the effects of transiently expressed proteins in a p o p u latio n w as the level and consistency of transfection. Thus, if only a
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