Barbara Ann Spruce

Small Molecule Antagonists of the σ-1 Receptor Cause Selective Release of the Death Program in Tumor and Self-Reliant Cells and Inhibit Tumor Growth in Vitro and in Vivo

The acquisition of resistance to apoptosis, the cell’s intrinsic suicide program, is essential for cancers to arise and progress and is a major reason behind treatment failures. We show in this article that small molecule antagonists of the σ-1 receptor inhibit tumor cell survival to reveal caspase-dependent apoptosis. σ antagonist-mediated caspase activation and cell death are substantially attenuated by the prototypic σ-1 agonists (+)-SKF10,047 and (+)-pentazocine. Although several normal cell types such as fibroblasts, epithelial cells, and even σ receptor-rich neurons are resistant to the apoptotic effects of σ antagonists, cells that can promote autocrine survival such as lens epithelial and microvascular endothelial cells are as susceptible as tumor cells. Cellular susceptibility appears to correlate with differences in σ receptor coupling rather than levels of expression. In susceptible cells only, σ antagonists evoke a rapid rise in cytosolic calcium that is inhibited by σ-1 agonists. In at least some tumor cells, σ antagonists cause calcium-dependent activation of phospholipase C and concomitant calcium-independent inhibition of phosphatidylinositol 3′-kinase pathway signaling. Systemic administration of σ antagonists significantly inhibits the growth of evolving and established hormone-sensitive and hormone-insensitive mammary carcinoma xenografts, orthotopic prostate tumors, and p53-null lung carcinoma xenografts in immunocompromised mice in the absence of side effects. Release of a σ receptor-mediated brake on apoptosis may offer a new approach to cancer treatment.

Endogenous opioids and oligodendroglial function: possible autocrine/paracrine effects on cell survival and development.

Previous work has shown that oligodendrocytes (OLs) express both μ‐ and κ‐opioid receptors. In developing OLs, μ receptor activation increases OL proliferation, while the κ‐antagonist nor‐binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin‐derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1–17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1–17) was undetectable. To assess the function of OL‐derived opioids, the effect of κ‐agonists/antagonists on OL differentiation and death was explored. κ‐Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that κ‐receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms. GLIA 35:156–165, 2001.

Ontogeny of proenkephalin mRNA and enkephalin peptide expression in the cerebellar cortex of the rat: spatial and temporal patterns of expression follow maturational gradients in the external granular layer and in Purkinje cells

Proenkephalin mRNA and peptide products were examined in developing cells of the postnatal rat cerebellar cortex using in situ hybridization and immunocytochemistry. On day 7, proenkephalin mRNA was first detected as discrete cellular labeling in Golgi cells and as a diffuse hybridization signal over the Purkinje cell layer. On day 14, proenkephalin mRNA and peptide products primarily appeared in distinct subpopulations of Purkinje cells present in the posterior and lateral cerebellum. Similarly, in the external granular layer (EGL), enkephalin immunoreactivity was present only in the posterior and lateral portions of the cerebellum on day 14. However, proenkephalin mRNA was not detected in enkephalin-immunoreactive EGL cells. On day 21, the subset of Purkinje cells that expressed proenkephalin mRNA and peptides were distributed more uniformly throughout the cerebellum. On day 28, a few enkephalin-immunoreactive Purkinje cells were uniformly present throughout the cerebellum, but proenkephalin mRNA was not detected in most of these cells. The spatial gradients in proenkephalin mRNA expression evident in the Purkinje cells of younger rats were no longer present in 28-day-old rats. These findings are important, because endogenous opioids such as enkephalin have been previously shown to inhibit the growth of Purkinje cell dendrites and dendritic spines, and inhibit the rate of mitosis in EGL neuroblasts. Cells do not develop at uniform rates within the cerebellum. There are regional differences in the timing of the formation of the EGL, and in the morphogenesis of Purkinje cells. In conjunction with previous work, the present findings suggest that during development, the pattern of enkephalin immunoreactivity in Purkinje and EGL cells closely follows the spatial and temporal gradients of maturation in both these cell types. The emergence and disappearance of enkephalin immunoreactivity in Purkinje and EGL cells is spatially and temporally related, and coincides with proenkephalin mRNA expression in Purkinje cells. Thus, the transient and coordinated appearance of enkephalin in cerebellar Purkinje and EGL cells may contribute to regional differences in the rate of cerebellar maturation, and may help synchronize the developmental interactions between these two cell types.