Protein degradation in human T-lymphocytes

Abstract

Protein from resting or phytohemagglutinin-stimulated human peripheral blood T-lymphocytes, pulse-labeled 'in vitro' for 1 h with 3H-leucine, had a half-life of 30 h.Small, nondividing lymphocytes have been used to study the activation process induced by mitogens because metabolic, functional and morphological parameters undergo large-scale changes 1. Therefore, these cells also provide a convenient model to clarify the regulation of protein metabolism, because protein synthesis can be regulated in a precise way during activation. Although there has been considerable work on protein synthesis in lymphocytes 1, little has been done in the area of protein degradation, a process which is of equal importance with synthesis in maintaining and regulating the protein content of the cells 2. The present paper describes the general characteristics of protein degradation in human T-lymphocytes. Material and methods.Human peripheral blood T-lymphocytes were obtained as previously described 3. Cell viability, checked by trypan blue exclusion, was over 95% and differential counts showed 96.0% T-cells. Cells were incubated at 37 ~ in an atmosphere of 5% CO 2 in RPMI 1640 medium (Flow Lag, Irvine, Scotland) (106 viable cells/ml) with 10% foetal bovine serum 100 U/ml penicillin and 100 gg/ml streptomycin. For phytohemagglutinin (PHA) stimulation, T-cells were incubated at 37 ~ for 30 h in the same medium containing 25 gg/ml PHA-P (Difco Lag, Detroit, Michigan, USA). To study the time course of the stimulation, T-lymphocytes cultures were incubated as described above for 6, 24, 48, 72 and 96 h. 1 h before ending the incubation period, cultures were pulse-labeled with [methyl-3H]-thymidine (2 ~tCi/ml, 2 Ci/mmole). Autoradiographs were made, as previously described 4, by the 'stripping film' technique. Labeled and unlabeled lymphocytes were evaluated in 3 smears per case (300 cells/case). Lymphocytes from untreated chronic lymphocytic leukaemia (CLL) diagnosed as T-cell leukaemia were isolated and incubated under the same conditions as the normal lymphocytes. For radioactive labeling, cells were incubated in medium 3 containing L-[4, 5-HI leucine (0.05 mM, 52 Ci/mmole) for 1 h in siliconized Erlenmeyer flasks at 37~ with shaking. Then, the cells were washed 4 times with fresh medium containing 2 mM unlabeled leucine with 5 min incubations at 37 ~ between resuspension of the cells in medium and centrifugations at 300 x g. After washing, the cells were chased at 37~ in fresh medium containing 2 mM L-leucine for 24 h. Aliquots of medium were removed at different intervals, cooled in ice and centrifuged (300x g, 5 min) at 4 ~ The cell pellet was resuspended in phosphate buffered saline. Supernatants and cells were precipitated with 5% trichloroacetic acid (TCA). Precipitates were dissolved in 0.2 N NaOH. Radioactivity was determined by liquid scintillation counting. All counts were corrected for quenching using an internal standard. The TCA-soluble radioactivity of supernatant and cells at each time-point was calculated as the differen...