Vol. 13, Issue 3, 947-964, March 2002

Proliferating or Differentiating Stimuli Act on Different Lipid-dependent Signaling Pathways in Nuclei of Human Leukemia Cells

Luca M. Neri,^* Roberta Bortul, Paola Borgatti,^* Giovanna Tabellini, Giovanna Baldini, Silvano Capitani,^* and Alberto M. Martelli^§

 ^*Dipartimento di Morfologia ed Embriologia, Sezione di Anatomia Umana Normale, Università di Ferrara, 44100 Ferrara, Italy;  Istituto di Citomorfologia Normale e Patologica del Consiglio Nazionale delle Ricerche, c/o Istituti Ortopedici Rizzoli, 40137 Bologna, Italy;  Dipartimento di Morfologia Umana Normale, Università di Trieste, 34138 Trieste, Italy;  ^§Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia, School of Pharmacy, Università di Bologna, 40126 Bologna, Italy

Previous results have shown that the human promyelocytic leukemia HL-60 cell line responds to either proliferating or differentiating stimuli. When these cells are induced to proliferate, protein kinase C (PKC)-II migrates toward the nucleus, whereas when they are exposed to differentiating agents, there is a nuclear translocation of the  isoform of PKC. As a step toward the elucidation of the early intranuclear events that regulate the proliferation or the differentiation process, we show that in the HL-60 cells, a proliferating stimulus (i.e., insulin-like growth factor-I [IGF-I]) increased nuclear diacylglycerol (DAG) production derived from phosphatidylinositol (4,5) bisphosphate, as indicated by the inhibition exerted by 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and U-73122 (1-[6((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), which are pharmacological inhibitors of phosphoinositide-specific phospholipase C. In contrast, when HL-60 cells were induced to differentiate along the granulocytic lineage by dimethyl sulfoxide, we observed a rise in the nuclear DAG mass, which was sensitive to either neomycin or propranolol, two compounds with inhibitory effect on phospholipase D (PLD)-mediated DAG generation. In nuclei of dimethyl sulfoxide-treated HL-60 cells, we observed a rise in the amount of a 90-kDa PLD, distinct from PLD1 or PLD2. When a phosphatidylinositol (4,5) bisphosphate-derived DAG pool was generated in the nucleus, a selective translocation of PKC-II occurred. On the other hand, nuclear DAG derived through PLD, recruited PKC- to the nucleus. Both of these PKC isoforms were phosphorylated on serine residues. These results provide support for the proposal that in the HL-60 cell nucleus there are two independently regulated sources of DAG, both of which are capable of acting as the driving force that attracts to this organelle distinct, DAG-dependent PKC isozymes. Our results assume a particular significance in light of the proposed use of pharmacological inhibitors of PKC-dependent biochemical pathways for the therapy of cancer disease.