A necessary role for reduced intracellular potassium during the DNA degradation phase of apoptosis.

Apoptosis is a highly organized mechanism that allows specific cells to die in a controlled manner. Apoptosis can be induced by a variety of agents in a great number of cell types, but cell shrinkage and discrete chromatin cleavage remain common components of apoptosis that is manifested by constitutive enzymes. Despite intensive investigation, the actual event or events that signal this cell death process have not been completely identified. Because of their ability to effect a great number of cellular enzymes simultaneously, we have hypothesized that ions in high concentration (150 mM) in living cells may act as a repressor of apoptotic effectors. In this manuscript we review data that shows that DNA degradation only occurs in the shrunken population of lymphocytes treated with glucocorticoid, shrunken cells loose K+ and become hypotonic, and physiological concentrations on K+ inhibit nuclease activity irrespective of proteins. Together, these data suggest that the reduction in potassium levels appears necessary for the DNA degradation that occurs during apoptosis.

Native recombinant cyclophilins A, B, and C degrade DNA independently of peptidylprolyl cis-trans-isomerase activity. Potential roles of cyclophilins in apoptosis.

Previous work in our laboratory (Montague, J., Gaido, M., Frye, C., and Cidlowski, J. (1994) J. Biol. Chem. 269, 18877-18880) has shown that human recombinant cyclophilins A, B, and C have sequence homology with the apoptotic nuclease NUC18 and that denatured cyclophilins can degrade DNA. We have now evaluated the nucleolytic activity of recombinant cyclophilins under native conditions. We show that nuclease activity inherent to cyclophilins is distinct from cis-trans-peptidylprolyl isomerase activity and is similar to that described for apoptotic nucleases. Cyclophilin nucleolytic activity is stimulated by Ca2+ and/or Mg2+, with a combination of the two being optimal for cyclophilins A and B. Mg2+ alone is sufficient for cyclophilin C nuclease activity. pH optimums are in the range of pH 7.5-9.5. Cyclophilins can degrade both single-stranded and double-stranded DNA. Additionally, cyclophilins produce 3'-OH termini in linear double-stranded substrates, suggesting the cuts produced are similar to those of apoptotic cells. Cyclophilins also display endonucleolytic activity, demonstrated by their ability to degrade supercoiled DNA. In the absence of ions, cyclophilins bind linearized DNA. When added to nuclei from nonapoptotic cells, cyclophilin C induces 50-kilobase pair DNA fragmentation but not internucleosomal fragmentation. Together, these data suggest that cyclophilins are involved in degradation of the genome during apoptosis.

Cellular catabolism in apoptosis: DNA degradation and endonuclease activation.

Recent research has focused on identifying the biochemical events associated with the apoptotic process. These include specific degradation of the chromatin which was described by Wyllie in 1980 [1], with the report of the appearance of discretely sized DNA fragments from apoptotic rat thymocytes. The fragments corresponded in size to strands of DNA that were cleaved at internucleosomal regions and create a 'ladder pattern' when electrophoresed on an agarose gel. Because of its near universality, internucleosomal DNA degradation is considered a diagnostic hallmark of cells undergoing apoptosis. It is of great interest to identify the enzymes involved, and some of the candidates will be discussed.

The biochemistry and molecular biology of glucocorticoid-induced apoptosis in the immune system.

Apoptosis is a form of programmed cell death that occurs under numerous developmental and physiological conditions that require the selective elimination of cells from tissues and organs without the production of an inflammatory response. The initiation of apoptosis is controlled by a regulation of the balance between death and life signals perceived by the cell. A typical response of cells to an apoptotic stimulus includes a reduction in cell volume, compaction of intracellular organelles, chromatin condensation, and the generation of apoptotic bodies which contain degraded cellular components. Apoptotic bodies are often engulfed by neighboring cells or macrophages, preventing the occurrence of an inflammatory response in the region of the dying cells. Although the molecular basis for this cellular suicide is poorly understood, evidence indicates that apoptosis is an active process, requiring energy for its effective completion. We have sought to define the catabolic "effector" molecules that carry out the apoptotic process using glucocorticoid-induced apoptosis in rodent and human lymphocytes as model systems. These cells respond to dexamethasone with an arrest of cell growth, chromatin condensation, cell shrinkage, and the selective degradation of DNA, RNA, and protein. These effects are dependent on the presence of functional glucocorticoid receptors and require gene expression. The fragmentation of DNA and its associated cell shrinkage has been a focus of our efforts, because these effects reflect an irreversible commitment to death. Accordingly, we have developed assays to study apoptosis at the single cell level and to identify, purify, and clone the nuclease(s) that cause DNA damage in apoptotic cells. Using these approaches, we have identified and characterized a novel low molecular weight nuclease (NUC18) whose activity correlates with the DNA degradation occurring during apoptosis. NUC18 requires calcium for optimal activity in vitro and is inhibited by zinc and aurintricarboxylic acid, two known inhibitors of apoptosis. The amino acid sequence of pure NUC18 reveals a surprising homology to the cyclophilin family of proteins. Furthermore, recombinant cyclophilins have biochemical and pharmacological properties identical to those of NUC18. We have also studied the molecular basis for the catabolism of RNA and proteins that occurs during lymphocyte apoptosis. Recent experiments have identified selective cleavage of 28S ribosomal RNA and a novel nonlysosomal protease, both of which contribute to the demise of the cell. In summary, we present an evolving model that unifies the activation of apoptosis in lymphocytes by glucocorticoids with the counter-balancing effect of inhibitors such as Bcl-2.

A calcium-dependent nuclease from apoptotic rat thymocytes is homologous with cyclophilin. Recombinant cyclophilins A, B, and C have nuclease activity.

Apoptosis is an important physiological process that involves the deletion of specific cells in a controlled and timely manner. A biochemical hallmark typifying apoptosis in normal lymphocytes is DNA cleavage caused by a calcium-dependent nuclease. We have previously identified and purified an 18-kDa nuclease (NUC18) from glucocorticoid-treated rat thymocytes whose activity is associated with this apoptotic DNA fragmentation. Partial protein sequencing of pure NUC18 has generated two peptide sequences that have a remarkable similarity to rat cyclophilin A and other members of the cyclophilin family. We report here that recombinant cyclophilins A, B, and C have a calcium/magnesium-dependent nuclease activity with biochemical and pharmacological properties similar to those of NUC18. Our results raise the intriguing possibility that cyclophilin or a cyclophilin-related protein may play a role in lymphocyte apoptosis.

Nonchromaffin paraganglioma (chemodectoma) of thyroid region.

A case of a paraganglioma located over the thyroid cartilage in the midline is reported. The tumor had all the appearances of a carotid body tumor, but its odd location could not be explained on the basis of previously known areas where paraganglionic tissues have been found. Since a paraganglioma has also hitherto been described within the thyroid gland, clinicians and pathologists alike are cautioned that unusually located paragangliomas may be expected in the future.