Numerous Investigations have documented the role of retinoids as biological modifiers of growth and differentiation. Many leukemias have been characterized as their primary defect a failure to carry through the normal differentiation of the cells. Retinoids, and other differentiating agents, have been employed clinically to try to restore the complete differentiation program and have been reported to possess therapeutic efficacy in a variety of proliferative disorders eg. preleukemic syndromes, acute leukemias and some solid tumors. The mechanisms by which retinoids perform these functions remain unclear. Clarification of the underlying biochemical mechanisms should allow for the generation of new and novel therapeutic approaches to these disorders as well as providing insight into the normal control processes in cellular differentiation. The addition of retinoic acid (RA) to the human promyelocytic leukemia cell line (HL60), as well as to blasts from patients with acute promyelocytic leukemia, results in the differentiation of the cells to the mature myeloid phenotype. Early during the differentiation process, and prior to phenotypic differentiation, there is a marked stimulation of a Ca2+ & phospholipid (PL) dependent protein kinase designated protein kinase C (PK-C) and the phosphorylation of specific endogenous proteins in a Ca2+ & PL dependent reaction. Phorbol esters very rapidly (minutes) reverse both increases in PK-C activity and protein phosphorylation but only slowly (days) inhibit control levels of these parameters seggesting that RA induction leads to the presence of an altered form of the enzyme. This proposal is concerned with delineating the role of PK-C in RA-induced myeloid differentiation and the mechanism of phorbol ester modulation of PK-C activity by: A) Investigating the correlation between PK-C and myeloid differentiation utilizing: 1. Conditions of syngergistic induction of differentiation with RA and agents raising intracellular cAMP. II. Promyelocytic cell lines resistant to the effect of RA. III. Reversal of myeloid pathway differentiation by agents which cause monotyte/macrophage differentiation ie. phorbol esters and 1,25 dihydroxycholecalciferol. B) Studying the mechanisms of changes in PK-C activity utilizing: I. Purification and characterization of the enzymes from control, RA induced and TPA treated cells. II. The preparation of monoclonal antibodies to study the synthesis, modification and cellular location of PK-C. III. Analysis of the subcellular localization of PK-C. C) Analysis of the specific cellular protein substrates for PK-C.
| Baciu, P C; Durham, J P (1990) A procedure for the extraction and high resolution two-dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei. Electrophoresis 11:162-6 |
| Spearman, T N; Fontana, J A; Butcher, F R et al. (1989) Effect of retinoic acid on the phosphorylation of endogenous proteins in HL-60 cells. J Cell Physiol 138:349-57 |
| Fontana, J; Miksis, G; Durham, J (1987) Elevation of adenylate cyclase activity during leukemic cell differentiation. Exp Cell Res 168:487-93 |
| Fontana, J A; Reppucci, A; Durham, J P et al. (1986) Correlation between the induction of leukemic cell differentiation by various retinoids and modulation of protein kinases. Cancer Res 46:2468-73 |
| Durham, J P; Emler, C A; Butcher, F R et al. (1985) Calcium-activated, phospholipid-dependent protein kinase activity and protein phosphorylation in HL60 cells induced to differentiate by retinoic acid. FEBS Lett 185:157-61 |
