Defects in cAMP-pathway may initiate carcinogenesis in dividing nerve cells: a review.

The mechanisms of carcinogenesis in nervous tissues are not well understood. It is now established that adenosine 3,',5'-cyclic monophosphate (cAMP)-pathway plays a crucial role in initiating differentiation in transformed and embryonic cells of neuronal and glial origin. Therefore, we propose that defects in the cAMP-pathway may initiate the first phase of carcinogenesis (immortalization). Subsequent genetic abnormalities in oncogenes, anti-oncogenes or other cellular genes individually or in combination may lead to transformation (cancer phenotype). This hypothesis is derived from the fact that an elevation of the cAMP level in murine NB cells induces terminal differentiation in many of these cells in spite of the fact that they are highly aneuploid. Additional changes in cAMP-regulated genes responsible for initiating differentiation may make these cells resistant to cAMP or may make the cAMP-effect on differentiation reversible. Indeed, cAMP-resistant cells exist in NB cell populations, and the cAMP-effect on differentiation is reversible in glioma cells. Identification of genes that initiate, promote and maintain terminal differentiation and those which prevent differentiation following elevation of cAMP in NB cells may increase our understanding of the mechanisms of carcinogenesis. This review illustrates the following: (a) historical background leading to the discovery of cAMP as an inducer of differentiation in nerve cells; (b) identification of potential sites in cAMP-pathway that may play a crucial role in initiating the first phase of carcinogenesis (immortalization) and potential gene targets in immortalized cells whose alterations may cause neoplastic transformation of nerve cells. It is interesting to note that the cAMP pathway remains responsive to an elevated cAMP level in inducing differentiation in NB cells in spite of chromosomal anomalies and genetic changes associated with the maintenance of a cancer phenotype.

Relative sensitivity of undifferentiated and cyclic adenosine 3',5'-monophosphate-induced differentiated neuroblastoma cells to cyclosporin A: potential role of beta-amyloid and ubiquitin in neurotoxicity.

Cyclosporin A is routinely used in transplant therapy following allogeneic or xenogeneic tissue transplantation to prevent rejection. This immunosuppressive drug is also neurotoxic; however, its mechanisms of action for neurotoxicity are poorly understood. Undifferentiated and cyclic adenosine 3',5'-monophosphate (cAMP)-induced differentiated neuroblastoma (NB) cells were used as an experimental model to study the toxicity of cyclosporin A. Results showed that cyclosporin A promoted the outgrowth of neurites and inhibited the growth of undifferentiated NB cells. When cyclosporin A was added simultaneously with RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, or with prostaglandin E1, a stimulator of adenylate cyclase, it markedly enhanced the growth inhibitory and differentiation effects of these cAMP-stimulating agents. In addition, cyclosporin A added to cAMP-induced differentiated NB cells caused dose-dependent degeneration of these cells as evidenced by the vacuolization of cytoplasm and the fragmentation of nuclear and cytoplasmic materials; however, neurites remained intact. Cyclosporin A alone did not alter the intensity of cell immunostaining for ubiquitin or beta-amyloid peptide (amino acids 1-14) (Abeta1-14); however, it enhanced the intensity of staining for both ubiquitin and Abeta in cells that were treated with cAMP-stimulating agents. The intensity of staining of amyloid precursor protein (amino acids 44-63) (APP44-66) did not change in any treated group, suggesting that the increase in Abeta staining is due to increased processing of APP to Abeta. We propose that one of the mechanisms of cyclosporin A-induced neurotoxicity involves increased levels of Abeta and ubiquitin.

Cyclosporin A regulates the levels of cyclophilin A in neuroblastoma cells in culture.

Cyclophilin A (CyP-A), a member of a highly conserved family of proteins, immunophilins, is the major intracellular receptor for the immunosuppressive drug, cyclosporin A (CsA). CyP-A is widely expressed in many tissues, but is found in the highest concentration in brain tissues and may perform critical neuronal functions. CsA is a known neurotoxin. Therefore, understanding the regulation of CyP-A levels in nerve cells, particularly by CsA, is important. We have utilized murine neuroblastoma (NB) cells as an experimental model to investigate this issue. Our results show that CsA alone was sufficient to induce morphological differentiation in undifferentiated NB cells and to increase CyP-A levels as determined by immunostaining. However, inducing terminal differentiation by elevating adenosine 3',5'-cyclic monophosphate (cAMP) levels using either 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724), an inhibitor of cyclic nucleotide phosphodiesterase, or prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, was not sufficient to increase CyP-A levels. CsA was required to increase CyP-A levels in both RO20-1724- and PGE1-induced differentiated NB cells. Increases in CyP-A levels, however, occurred without any change in the expression of the CyP-A gene as determined by reverse-transcriptase polymerase-chain reaction analysis using (CyP-A)-specific primers. These results suggest that CsA regulates the level of its own binding protein, CyP-A, in both undifferentiated and cAMP-induced differentiated NB cells in culture.

Prostaglandins act as neurotoxin for differentiated neuroblastoma cells in culture and increase levels of ubiquitin and beta-amyloid.

Although chronic inflammatory reactions have been proposed to cause neuronal degeneration associated with Alzheimer's disease (AD), the role of prostaglandins (PGs), one of the secretory products of inflammatory reactions, in degeneration of nerve cells has not been studied. Our initial observation that PGE1-induced differentiated neuroblastoma (NB) cells degenerate in vitro more rapidly than those induced by RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, has led us to postulate that PGs act as a neurotoxin. This study has further investigated the effects of PGs on differentiated NB cells in culture. Results showed that PGA1 was more effective than PGE1 in causing degeneration of differentiated NB cells as shown by the cytoplasmic vacuolation and fragmentation of soma, nuclei, and neurites. Because increased levels of ubiquitin and beta-amyloid have been implicated in causing neuronal degeneration, we studied the effects of PGs on the levels of these proteins during degeneration of NB cells in vitro by an immunostaining technique, using primary antibodies to ubiquitin and beta-amyloid. Results showed that PGs increased the intracellular levels of ubiquitin and beta-amyloid prior to degeneration, whereas the degenerated NB cells had negligible levels of these proteins. These data suggest that PGs act as external neurotoxic signals which increase levels of ubiquitin and beta-amyloid that represent one of the intracellular signals for initiating degeneration of nerve cells.

Insulin regulation of lipoprotein lipase in cultured isolated rat adipocytes.

The cellular regulation of adipose tissue lipoprotein lipase by insulin was investigated using cultured isolated rat adipocytes. Evidence for sustained cell viability over 3 days included stability of specific [125I]insulin binding and adipocyte number. Lipoprotein lipase was measured in three functional compartments: 1) enzyme activity secreted into the culture medium, 2) activity releasable from cell suspensions by heparin, and 3) activity extractable from cells (after maximal heparin release) in deoxycholate and detergent. One day after preparation, these activities stabilized and were 1.3 +/- 0.2, 1.4 +/- 0.2, and 7.7 +/- 0.9 neq/10(6) cells X min, respectively (n = 24, mean +/- SEM). Insulin, added the day after preparation, produced a dose-dependent (1-400 ng/ml) increase in lipoprotein lipase releasable from cells by heparin at 2, 4, and 24 h. Insulin also increased intracellular enzyme measured as deoxycholate-detergent-solubilized activity extracted from previously heparin-released cells. However, insulin-mediated increases in culture medium enzyme only occurred subsequent to cellular effects. All insulin-mediated effects were prevented by cycloheximide (1 microgram/ml). Thus, insulin increased two cellular pools of adipocyte lipoprotein lipase in a dose-dependent manner, but had no direct effect on enzyme secretion. Overall, cultured isolated rat adipocytes appear to be a valuable system for the study of lipoprotein lipase regulation at the level of the adipocyte.