Staurosporine analogs promote distinct patterns of process outgrowth and polyploidy in small cell lung carcinoma cells.

We have recently shown that staurosporine mediates the conversion of small cell lung carcinoma (SCLC) cells into a neuron-like process-bearing phenotype. Here, we have extended these studies to the staurosporine analogs K252a, lestaurtinib, PKC412, stauprimide, and UCN-01 and analyzed their influence on process extension, cell cycle distribution, and induction of polyploidy in four SCLC cell lines. In GLC-2 cells, all compounds provoked extensive process formation with the exception of PKC412 that showed no response. In H1184 cells, process formation was predominantly induced by staurosporine and, to lesser extent, in lestaurtinib-, stauprimide-, and UCN-01-treated cells. In the presence of K252a or PKC412, cells became bipolar and spindle shaped or showed pronounced cell flattening. In GLC-36 and SCLC-24H cells, only cell flattening was detectable. Process formation was reversible upon drug removal as shown for GLC-2 and H1184 cells. Fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) analysis indicated the induction of polyploidy in all staurosporine and in two out of four stauprimide-treated SCLC cell lines. For other staurosporine analogs, polyploidy was observed only in UCN-01-treated GLC-36 cells and in K252a-treated H1184 and GLC-36 cells. The presence of staurosporine or its analogs did not alter the constitutive activation pattern of the canonical Akt/PI3K or MEK/extracellular signal-regulated kinase (ERK)1/2 signaling pathways nor could we detect an influence of stauprimide application on the expression level of the c-Myc oncogene. These data demonstrate that in SCLC cells, albeit a higher substrate specificity, staurosporine analogs can induce staurosporine-comparable effects.

Regulation of the NPC2 protein-mediated cholesterol trafficking by membrane lipids.

Recycling and turnover of cell membranes play a critical role in cell metabolism. The internalization of membranes through the different processes of endocytosis, phagocytosis, and autophagy deliver a considerable amount of membranes and lipids to the endosomal and lysosomal system which is tasked with its degradation. Its failure to do so leads to severe fatal neurodegenerative diseases. In order to better understand how membranes are degraded, we have to investigate the complex interactions that take place in this compartment between complex membrane lipids, enzymes and lipid binding and transfer proteins involved. To this end, we developed lipid transfer and fusion assays which allow us to quantify these interactions and assess their specificity. The published results of these investigations are summarized in this article. One of our main conclusions is that we have provided evidence for the hypothesis that acid sphingomyelinase stimulates Niemann pick disease protein type 2-mediated cholesterol export substantially by converting sphingomyelin to ceramide in the inner membranes of late endosomes.

Biological function of the cellular lipid BMP-BMP as a key activator for cholesterol sorting and membrane digestion.

Bis(monoacylglycero)phosphate (BMP) is one of the most intriguing classes of lipids discovered to date. Its biosynthetic pathway is still not fully known, even though studies point to cardiolipin and PG as sources. BMP has an unusual stereoconfiguration which is responsible for its slow catabolism. It is localized exclusively in the inner membranes of late endosomes, and mostly, lysosomes. BMP is negatively charged at the acidic pH of lysosomes, and these charges are central to its role in the degradation of lipids and membranes in the lysosome. In effect, these negative charges facilitate the adhesion of the soluble positively charged hydrolases and activator proteins, thus allowing them to degrade the lipids at the interface of the inner membranes of the lysosome. In this review, we will summarize the different properties of BMP and showcase its importance in the catabolism of lipids and membranes in the lysosome.

Optimization of submerged keratinocyte cultures for the synthesis of barrier ceramides.

Epidermal differentiation results in the formation of the extracellular lipid barrier in the stratum corneum, which mainly consists of ceramides, free fatty acids, and cholesterol. Differentiating keratinocytes of the stratum granulosum synthesize a series of complex long-chain ceramides and glucosylceramides with different chain lengths and hydroxylation patterns at intracellular membranes of the secretory pathway. Formation of complex extracellular ceramides parallels the transition of keratinocytes from the stratum granulosum to the stratum corneum, where their precursors, complex glucosylceramides and sphingomyelin, are secreted and exposed to extracellular lysosomal lipid hydrolases. Submerged cultures used so far showed a reduced ceramide content compared to the native epidermis or the air-exposed, organotypic culture system. In order to investigate the sphingolipid metabolism during keratinocyte differentiation, we optimized a simple cell culture system to generate the major barrier sphingolipids. This optimized model is based on the chemically well-defined serum-free MCDB medium. At low calcium ion concentrations (0.1mM), keratinocytes proliferate and synthesize mainly Cer(NS) and a small amount of Cer(NP). Supplementation of the MCDB cell culture medium with calcium ions (1.1mM) and 10 microM linoleic acid triggered differentiation of keratinocytes and synthesis of a complex pattern of free and covalently bound ceramides as found in native epidermis or air-exposed organotypic cultures, though at a reduced level. The mRNA levels of the differentiation markers keratin 10 and profilaggrin increased, as well as those of ceramide glucosyltransferase and glucosylceramide-beta-glucosidase. The described culture system was thus suitable for biochemical studies of the sphingolipid metabolism during keratinocyte differentiation. The addition of serum or vitamin A to the medium resulted in a decrease in ceramide and glucosylceramide content. Lowering the medium pH to 6, while maintained cell viability, led to an increase in the processing of probarrier lipids glucosylceramide and sphingomyelin to free ceramides and protein-bound ceramide Cer(OS).

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis.

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.

Nitric oxide regulates synthesis of gene products involved in keratinocyte differentiation and ceramide metabolism.

During terminal differentiation of keratinocytes the expression of various proteins, which are required for the formation of the epidermal water barrier in the skin of land dwelling animals, is upregulated. Using a cell culture model for the differentiation of human keratinocytes and real-time PCR, we quantified the mRNA levels of several proteins involved in differentiation and ceramide metabolism. A calcium shift (1.1 mM CaCl2, 10 microM linoleic acid) for 8 days triggered an increase in mRNA levels of keratin 10 (75-fold), profilaggrin (55-fold), glucosylceramide synthase (40-fold), beta-glucocerebrosidase (30-fold), prosaposin (15-fold), acid sphingomyelinase (5-fold), and serine palmitoyltransferase (SPTLC2, 4-fold). However, mRNA levels of keratin 14 and acid ceramidase did not change significantly. On the other hand nitric oxide added at concentrations lower than 0.25mM stimulates proliferation of keratinocytes (Krischel et al., J. Invest. Dermatol. 111, 286-291, 1998). Accordingly, the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.2 mM) had no effect on the morphology of cultured keratinocytes, whereas in cultured human fibroblasts apoptosis was induced. The expression patterns obtained suggest that keratinocytes remain in a basal proliferative state, with a 3-fold increase in keratin 14 expression, a marked decrease in mRNA levels of differentiation markers and of most ceramide-metabolizing enzymes to negligible levels. The inhibitor of the NO synthase, N(G)-nitro-L-arginine-methyl ester (L-NAME, 10 mM), induced a transient increase in ceramide formation, followed by apoptosis in keratinocytes but not in fibroblasts. Both, SNAP and L-NAME, decreased the mRNA levels of all proteins involved in ceramide metabolism.