DISCUSIÓN Y RESULTADOS.
4.2.3.2 PRINCIPIO DE INTERCULTURALIDAD a Ley 29785 Ley del Derecho a la Consulta.
the relationship between p21ras and PKC in T cell activation.
Two pieces of evidence suggested that p21ras was capable of having a role in T cell activation - first, it had been know n for some tim e that activated m utants of ras, in cooperation w ith other oncogenes, such as myc or pim-1, could transform lym phocyte lineages from transgenic mice(250). This transform ed phenotype suggests that ras m ight be able to contribute to gene expression in lymphocytes, thereby overriding the need for external grow th stim uli. IL2 is the m ajor T cell grow th factor, and so one possible explanation for the transform ing effect of Ras could be its ability to control IL2 gene expression, over-riding the need for external signals. Indeed, ras has becom e know n as a signalling 'sw itch' m olecule, c o n stitu tiv ely activated m utants being thought to constantly deliver an on' signal that m ay preclude the need for grow th or activation signals(179, 180). More recently, it has been observed th at u p o n activation of n o rm al h u m an peripheral blood lym phocytes and various T cell lines, the p21ras proteins are rapidly activated for prolonged periods of time(4). Further experim ents dem onstrated that pharm acological stim uli that w ere know n to contribute to the activation of the IL2 gene, a m arker for T cell activation, also w ere pow erful stimuli for the activation of p21ras.
In lym phocytes, T cell activation defines the biochemical events occurring after TCR lig atio n w hich cu lm in ate in th e activ atio n of IL2 gene
transcription and IL2 secretion(8). The physiological trigger for IL2 gene induction is provided by ligation of the TCR. TCR ligation occurs w hen a T cell encounters antigen in the context of MHC on the surface of an antigen presenting cell(APC). (lO)For experim ental purposes, TCR ligation can be achieved by cross linking the TC R /C D 3 com plex w ith antibodies; the requirem ent for TCR triggering can be bypassed altogether by the use of phorbol esters, w hich activate protein kinase C, and calcium ionophores, w hich elevate intracellular calcium concentration(251). IL2 gene activation can be m onitored using a bioassay, m easuring the levels of IL2 secreted into culture supernatant. As described in M aterials and M ethods, supernatants are tested for their ability to sup p o rt the grow th of a cell line absolutely dependent on exogenous IL2 for proliferation. The signals that regulate IL2 gene activity have been u nder study for m any years, and thus m uch data has been accumulated. Table 3.1 illustrates typical IL2 production in
Table 3.1 IL2 produ ctio n in T cells.
stim u li EL4 p e r i p h e r a l b l o o d lym phocytes io n o - - PdBu + + ^ - TCR + + + iono+ pdbu + + + + 4-4- TCR+pdbu + + + + + + 3+ = < 5U /m l; ++ = 5- 20U/m l; +++ = > 20U /m l
res p o n se to v a rio u s ag en ts in n o rm al h u m a n p e rip h e ra l b lo o d lym phocytes, and a m urine T cell line, EL4. Thus, PBLs do not produce detectable quantities of IL2 in the presence of only calcium ionophore, phorbol esters, or TCR antibodies(62); it seems that at least tw o 'types' of
stim ulating agents m ust be applied in order to detect activation of the IL2 gene in these cells. These activating signals are PKC activated w ith phorbol esters com bined w ith a calcium signal or TCR triggering. EL4 cells, w hich have also been used to study the regulation of the IL2 gene, do not produce IL2 in response to ionom ycin alone. In contrast, phorbol esters or TCR antibody alone are sufficient to stim ulate some IL2 secretion in EL4(48, 239). H ow ever, m axim al activation still requires the com bination of stim uli, as for PBLs.
The TCR and phorbol esters can both activate the p21ras proteins in T cells(4). Therefore, in this project I w ished to introduce dom inant m u tan t p21ras proteins into T cells and assess their effects upon IL2 gene activation. In particular, I w ished to exam ine w hether an activated m u tan t of Ras w ould be able to replace or substitute for TCR or PdBu signals for IL2 gene induction. Oncogenic v-H a-ras contains two am ino acid changes in the protein sequence at codons 12(G"^R) and 59(A ^ T)(252), w hich creates a protein insensitive to the effects of GAPs, and is oncogenic(253). Similarly, I w anted to assess w hether a dom inant inhibitory m utant of Ras w ould be able to block TCR and PdBu signals. Thus, another Ras m utant, N17ras, contains a single amino acid-substitution - S ^ R at codon 17 - which creates a Ras protein that cannot associate w ith effector proteins even w hen GTP loaded(226), and functions by com peting w ith endogenous Ras for guanine nucleotide exchange factors(186, 254). These tw o p roteins w ere used in transfection studies to investigate the affects and requirem ents for Ras in T cell activation signalling pathw ays.
Two m ethods for investigating the role of p21ras in T cell activation w ere considered - first, I explored m aking stably transfected T cell lines w ith m utant Ras proteins. This approach was not used, as our m ain interest was to investigate the prim ary effects of Ras proteins in T cell activation and to avoid the complications of secondary effects due to transform ation. Second, I studied the possibility of transiently co-expressing m u tan t Ras proteins w ith IL2 rep o rter genes in T cells. This m ethod p ro v ed am enable to m a n ip u la tio n , an d w as u sed for all su b seq u e n t stu d ies. T ran sien t transfection can be accom plished by m any m eans, b u t electroporation, using the Bio Rad Gene Puiser, proved consistent and reliable. Initially, I had hoped to be able to study the effects of m utant Ras proteins in norm al h u m a n p e rip h e ra l blood lym phocytes, b u t m any a ttem p ts, v ary in g d ifferen t co n d itio n s, d id n o t resu lt in rep ro d u c ib le re p o rte r gene expression. Effort was then focused on the hum an T lym phom a Jurkat. For all of the initial studies, the effect upon IL2 gene transcription w as the m ain goal, and so secretion of IL2 w as to be used as an internal control for a c tiv a tio n . U n fo rtu n a te ly , a lth o u g h all Ju rk a t w ere re p ro d u c ib ly transfectable, none proved able to produce IL2 upon stim ulation. As almost a last resort, the m urine thymom a EL4 was tested for its ability to produce lym phokine, and was show n to make easily detectable quantities of IL2. As has been show n(table 3.1), the regulation of lym phokine production in EL4 is som ew hat different from w hat has been described for norm al T cells, in that stim ulation w ith phorbol esters alone is sufficient to cause the cells to produce IL2. Thus, in figure 3.1, EL4 cells stim ulated w ith the phorbol ester phorbol-12,13,-dibutyrate(PdBu) alone produce ~ 10pg/m l IL2, as com pared to h u m an PBLs w hich produce alm ost lOx less th an th at am ount w hen stim ulated w ith PdBu alone. EL4 cells thus fulfilled the criteria of a good