Protein Synthesis by Ribosomes from Blood Lymphocytes of Normals and Patients with Chronic Lymphocytic Leukemia (CLL)

Abstract

Human blood lymphocytes incubated under standard cell culture conditions synthesize only small amounts of RNA and protein and remain in a resting, non-dividing state. Under the influence of several mitogens such as phytohemagglutinin (PHA), lymphocytes increase their synthesis of RNA and protein, replicate DNA, and divide. Several investigators have demonstrated that the blood lymphocytes of chronic lymphocytic leukemia (CLL) respond poorly to in vitro stimulation by PHA or specific antigens, showing a delay in the appearance of increased macro-molecular synthesis and a less than maximal response compared to lymphocytes from normal controls (1, 2). The decreased capacity of CLL lymphocytes to undergo transformation may be responsible in part for the immunologic deficits noted clinically in this disease.

One feature of lymphocyte transformation that has not been previously examined is the intrinsic ability of ribosomes isolated from lymphocytes to catalyze protein synthesis. We have developed a cell-free protein synthesizing system capable of incorporating significant amounts of ¹⁴C-amino acids into protein and have used this system to study ribosomal activity in normal and CLL lymphocytes both before and after PHA stimulation.

Venous blood was drawn into a heparinized syringe and allowed to sediment. Separation of lymphocytes from other blood elements was achieved by passage of white cell-rich plasma diluted with an equal volume of Eagle's minimal essential medium through a column of washed nylon fiber (3). The usual yield was 6-8 × 10⁸ cells (> 98% lymphocytes) from 500 ml of normal blood. The patients with CLL were untreated individuals with white blood cell counts between 15,000 to 120,000/mm³. Ribosomes were immediately isolated from a portion of the cells. The remainder of the cells were cultured with PHA for 72 hr (4) after which the ribosomes were isolated.