Cyclic AMP/PKA-promoted apoptosis: insights from studies of S49 lymphoma cells.

Increases in cyclic AMP (cAMP) are pro-apoptotic in numerous cell types, but the mechanisms of cAMP-promoted apoptosis are poorly defined. We have used murine S49 T-lymphoma cells as a model to provide insight into these mechanisms. Increases in cAMP in wild-type (WT) S49 cells were first noted to kill these cells in the 1970 s, but only in recent years, it was shown that this occurs by the intrinsic (mitochondria-dependent) apoptotic pathway. The apoptotic response does not occur in protein kinase A-null (kin-) clonal variants of WT S49 cells and thus is mediated by protein kinase A (PKA). A second S49 clonal variant, cAMP-Deathless (D-), has PKA activity but lacks cAMP-promoted apoptosis. Apoptosis in WT S49 cells occurs many hours after cAMP/PKA-promoted G1 cell cycle arrest and involves increased expression of Bim, a pro-apoptotic member of the Bcl-2 (B-cell lymphoma-2) family. This increase in Bim expression does not occur in kin- or D- S49 cells and knockdown of Bim blunts cAMP-mediated apoptosis in WT cells. Cytotoxic T lymphocyte antigen-2 also appears to contribute to cAMP/PKA-promoted apoptosis of S49 cells. Based on time-dependent differences in gene expression between WT, D- and kin- S49 cells following incubation with 8-(4-chlorophenylthio)-cAMP, additional genes and proteins are likely involved in this apoptosis. Studies with S49 cells should reveal further insight regarding the mechanisms of cAMP/PKA-promoted cell death, including the identification of proteins that are targets to enhance (e. g., in cancer) or inhibit (e. g., cardiac failure) apoptosis in response to hormones, neurotransmitters, and drugs.

Measurement of enzyme activity.

To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.