Akt/PKB is a Ser/Thr protein kinase impli cated in inhibition of apoptosis and stimulation of cellular growth in several tissues by mechanisms including phos phorylation of the pro apoptotic proteins Bad and Bax, and suppression of pro apoptotic proteins such as Bim and PUMA, through phosphorylation of the forkhead path way . favouring the anti apoptotic effect of Mdm2 on p53 . and inhibition of cleavage of Bid protein. The aim of this study was to investigate the connection of the death receptor stimulation with the intrinsic pathway in the apoptosis of the type II cells RA FLS, and to analyse the possible relation between constitutively activated phospho inositol 3 kinase/Akt and the mechanisms of resis tance to Fas mediated apoptosis.

Materials and methods Fibroblast like synoviocytes FLS from 11 patients with RA were obtained at the time of synovectomy or total joint replacement. All RA patients fulfilled the American College of Rheumatology 1997 cri teria for RA classification. All patients gave informed, written consent. The study was performed according to the recommendations of the Declaration of Helsinki and with the approval of the Comit�� Etico de Investigaci��n Cl��nica de Galicia. Synovial tissue was minced and incubated with 10 ug/ml collagenase in serum free DMEM for three hours at 37 C. After diges tion, FLS were filtered through a nylon cell strainer, washed exten sively with DMEM, and cultured in DMEM supplemented with 10% v/v FCS, 1% penicillin streptomycin and 1% L glutamine in a humidified 5% carbon dioxide atmosphere.

Adherent cells were trypsinized and splited in a 1 3 ratio once the cells were 80 to 90% confluent. FLS from passages three to eight were used. Small interfering RNA transfection in FLS Bid small interfering RNA, a pool of four target specific 19 nucleotide siRNAs, and non silence control siRNA, a pool of four non targeting siRNAs, were pur chased from Dharmacon. siRNA transfections were performed as described elsewhere. Briefly, RA FLS at 80 to 90% confluence were transiently transfected with siRNA in Opti MEM I using 1. 25 ug/ml DharmaFECT 1. Bid suppression was analysed by western blot. Experiments were performed 48 hours after transfections. pDsRed2 Bid Vector transfection in FLS pDsRed2 Bid Vector, a 5. 3 Kb Carfilzomib mammalian expression vec tor that encodes a fusion of Discosoma sp red fluorescent protein and Bid, and the empty pDsRed2 vector, were purchased from Clontech.

RA FLS at 60% confluence were transiently transfected with 0. 5 ug pDsRed2 Bid vector or pDsRed2 vector in Opti MEM I using 4 ug/ml Lipofectamine and 9 ug/ml Plus Reagent. Bid expression was analysed by western blot and immunofluorescence assays. Experiments were performed 48 hours after transfections. Apoptosis and cell death assays RA FLS were cultured in 96 well plates with DMEM and 5% FCS.