Françoise Miot

A mechanism in the control of intracellular CAMP level: the activation of a calmodulin-sensitive phosphodiesterase by a rise of intracellular free calcium

The intracellular concentration of cAMP results from the balance between its synthesis by adenylate cyclase and its catabolism by the system of cyclic nucleotide phosphodiesterases. Positive regulation of the cAMP system by activation of adenylate cyclase has been the focus of most of the research on the regulation of the level of this nucleotide. With regard to negative controls, a direct inhibition of adenylate cyclase by a-adrenergic, cholinergic agonists and several peptides is now well characterized and accounts for multiple negative controls of cAMP generation (Jakobs et al., 1980). It is thus often assumed now that intracellular concentration of cAMP is only controlled at the level of its synthesis. Such a concept is an oversimplification as it neglects the potential of controls at the level of cAMP catabolism. Phosphodiesterases are rather complex enzymes consisting of multiple soluble and particulate forms (reviewed by Beavo et al., 1982). It has been demonstrated that they are distinct non-intercon-

A new tool for efficient transfection of dog and human thyrocytes in primary culture

The introduction of exogenous DNA into mammalian cells is commonly used to study the functions of gene products. However cells in primary culture are usually refractory to most transfection systems. Here we investigated the ability of a new lipid formulation, FuGENE 6 transfection reagent, to promote DNA uptake into dog and human thyroid cells in primary culture. Gene transfer was monitored by the expression of a Green Fluorescent Protein (GFP) reporter gene. We report that FuGENE 6 is particularly suited for the transfection of thyroid cells and does not interfere with their normal growth. Optimization of the experimental conditions, such as DNA amount, DNA/lipid ratio, cell density and incubation with the transfection mixture, was achieved by evaluating the percentage of GFP-expressing cells by FACS analysis. FuGENE 6 allowed us to obtain 8-15% thyrocytes expressing the reporter gene which represents an efficiency 100-fold superior to other transfection methods.

Loss of several cell functions including okadaic acid-induced apoptosis after multiple passages in FRTL-5 cells.

In FRTL-5 cells, cultured over a period of more than 3 years, different properties of the cells have been observed to undergo spontaneous changes in the course of aging, i.e. after an increase in the number of passages. This consists mainly in alterations in their morphological phenotype and in some of their functional properties. The morphology of the cells displayed a progressive disruption of the monolayer organization with a loss of cell-cell contacts and a marked rounding-up of the cells. The uptake of iodide was not modified nor was the expression of thyroglobulin (Tg) mRNA as determined at various time intervals in the course of the cells culturing. Estimation of the proliferation by counting the frequency of [3H]thymidine labeled nuclei revealed an age-related decline in the sensitivity to TSH mitogenic action associated with a reciprocal increase in the insulin synergistic effect. Aged cells (+/- 40 passages) lost their apoptosis sensitivity to the phosphatase inhibitor, okadaic acid (OA) but not to cycloheximide (CHX) and/or actinomycin D (act. D) exposure. Altogether these observations favor the existence of a shift towards transformed properties with only partial loss of differentiated functions.