ABSTRACT
Temporal variations in the expression of the ras oncogene, and its protein product, were investigated during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukaemic (MEL) cells. We highlight the fact that when comparisons were made between untreated, proliferating cells and HMBA-treated, differentiating cells using only one time-point, differences, both for the expression of the gene and the protein, were in most cases insignificant; standard deviations were high and the interpretation could be made that HMBA had little effect. Such interpretation fails to take account of the dynamic nature of the system, with single time-point studies giving incomplete information, which can be misleading. Multiple time analyses showed clearly that rhythmic patterns of expression were modulated by the differentiating agent. Time-dependent changes in the expression of mRNA specific to H- ras and N- ras, as well as in the expression of the Ras protein, when measured over periods of minutes or hours, were apparent. HMBA affected frequency and phasing of the rhythms. Regulation of the dynamics in this way may be crucial to the control of cell function and transformation.
Subject(s)
Cell Differentiation , Genes, ras/genetics , ras Proteins/metabolism , Acetamides/pharmacology , Animals , Antineoplastic Agents/pharmacology , Blotting, Northern , Blotting, Western , Cell Division , Leukemia, Erythroblastic, Acute , Mice , Models, Statistical , Periodicity , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Research Design , Time Factors , Tumor Cells, CulturedABSTRACT
Determinations of mRNA and enzyme protein analyses for the protein kinase C (PKC) isoforms, alpha, epsilon and zeta, representing the classical, novel and atypical groups, respectively, in murine erythroleukaemic (MEL) cells revealed the occurrence of cyclic behaviour in expression. Such rhythmicity was not apparent when multiple analyses using only a single time-point were considered; the importance of incorporating a time component into experimental design was demonstrated. On induction of differentiation over a period of five days with hexamethylene bisacetamide, changes in the patterns of expression of mRNA and protein were apparent, as compared with those of untreated, proliferating cells. Modulation of the dynamics of the various PKC isoforms may represent a regulatory mechanism for the control of a diversity of cellular functions.
Subject(s)
Cell Differentiation , Isoenzymes/genetics , Periodicity , Protein Kinase C/genetics , Acetamides/pharmacology , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Isoenzymes/analysis , Isoenzymes/immunology , Leukemia, Erythroblastic, Acute , Mice , Models, Statistical , Polymerase Chain Reaction , Protein Kinase C/analysis , Protein Kinase C/immunology , Protein Kinase C-alpha , Protein Kinase C-epsilon , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time Factors , Tumor Cells, CulturedABSTRACT
The initiation of receptor-mediated small artery contraction is dependent on inositol 1,4,5-trisphosphate-stimulated release of stored calcium. The role of the other product of inositol lipid hydrolysis, 1,2-diacylglycerol, in maintaining contraction remains controversial. Therefore, we have determined the contractile response of rat subcutaneous small arteries (< 300 microns i.d.), when mounted as ring preparations in a myograph, to noradrenaline, angiotensin II, KCl-induced membrane depolarization, and a cell-permeable diglyceride, dioctanoylglycerol. In parallel experiments, the conversion of this diglyceride to dioctanoylphosphatidate was studied in 32P-labeled vessels. Dioctanoylglycerol produced a slow-onset sustained contraction that was dependent on extracellular calcium. This was accompanied by the generation of the lipid dioctanoylphosphatidate. Noradrenaline and KCl induced rapid-onset sustained contractions and increased the production of dioctanoylphosphatidate (75% and 91%, respectively). In addition, dioctanoylglycerol levels were reduced (41%) after noradrenaline stimulation, suggesting activation of diacylglycerol kinase. In contrast, the contractile response to angiotensin II was transient, and this agonist did not significantly affect the conversion of dioctanoylglycerol to phosphatidate. Noradrenaline markedly increased (fourfold) the formation of endogenous phosphatidate, whereas endogenous 1,2-diacylglycerol was increased (47%) with angiotensin II. These results demonstrate that phosphatidate formation is regulated by vasoconstrictor hormones during receptor-mediated contraction, independent of diglyceride mass. Modulation of the levels of lipid second messengers downstream from phospholipid hydrolysis may represent a mechanism by which agonists that act through the same signaling system produce different contractile responses.
