The DNA synthesis of leukemic (L 2C) guinea pig B lymphocytes involves a permanent activation of protein kinase C without corresponding phosphoinositide hydrolysis

Abstract

L 2C B lymphocytes have a constant high DNA synthesis due to their continuous proliferative state. The addition of polymyxin B (PmB), a rather selective inhibitor of protein kinase C, stopped ( 3H)thymidine incorporation with an IC 50 of 10 μM when added 18 h before measuring DNA synthesis. Interestingly, PmB inhibition of DNA synthesis was suppressed when 4 nM 12- O-tetradecanoylphorbol-13-acetate was added along with PmB, indicating that PmB may act through inhibition of protein kinase C. In the node and spleen lymphocytes of normal guinea pigs, protein kinase C activity was entirely cytosolic and was eluted at 0.12 M NaCl when adsorbed on DEAE-cellulose. In L 2C leukemic lymphocytes, total protein kinase C activity was of the same order of magnitude, but 20% of it was associated with the membrane fraction. The lipid-dependent activity, eluted at 0.12 M NaCl from cytosolic and membrane fractions, was suppressed by staurosporine with an IC 50 of 10–40 nM and by polymyxin B with an IC 50 of 2–6 μM. Phosphoinositide metabolism was studied in the transformed cells. Incorporation of 32Pi into polyphosphoinositides was considerable, whereas much more time was required for a tiny incorporation of inositol. We detected no release of radioactive inositol triphosphate. Taken together, these results suggest that protein kinase C function is indispensible for triggering L 2C leukemic lymphocyte proliferation. The causes of this permanent activation merit further investigation.