PHOSPHORYLATION OF RETINOBLASTOMA SUSCEPTIBILITY GENE PROTEIN ASSAYEDIN INDIVIDUAL LYMPHOCYTES DURING THEIR MITOGENIC STIMULATION

Phosphorylation of the protein encoded by retinoblastoma susceptibilit y gene (pRb) is the key event of the cell cycle committing the cell to enter S phase and also required for progression through S and G(2). W e describe a new methodology to monitor pRb phosphorylation in individ ual cells and correlate it with the cell cycle position. Specifically, pRb phosphorylation in human lymphocytes was assayed immunocytochemic ally using mAb which recognizes underphosphorylated pRb (pRb(P-)) conj ugated with a fluorochrome of one color combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRb(T)) tagged with another color fluorochrome. DNA was stained with still another c olor fluorochrome and cell fluorescence was measured by multiparameter flow cytometry. Specificity of anti-pRb(P-) mAb was confirmed by prei ncubation of the permeabilized cells with phosphatase. Analysis of pRb (P-) or a ratio of pRb(P-)/pRb(T) revealed that pRb was underphosphory lated in over 98% of the nonstimulated lymphocytes. The proportion of cells with underphosphorylated pRb dropped to 20% between 3 and 8 h af ter addition of the mitogen phytohemagglutinin (PHA). Phosphorylation of pRb within a cell was rapid and complete since reactivity of indivi dual lymphocytes with anti-pRb(P-) mAb was lost abruptly rather than s tep-wise during stimulation. Phosphorylation of pRb coincided with the appearance of cyclin D3, which was induced 3 h and peaked 12 h after addition of PHA. The nonspecific protein kinase inhibitor staurosporin e at a concentration known to arrest lymphocytes in G(1) but not to in terfere with the induction of cyclin D3 (20 nM) prevented pRb phosphor ylation. The present assay can be applied for screening antitumor drug s targeting CDKs and be useful for monitoring pRb phosphorylation in h uman tumors, the feature of a possible prognostic value in oncology. ( C) 1998 Academic Press.