Insulin up-regulates a Glc3Man9GlcNAc2-PP-Dol pool in capillary endothelial cells not essential for angiogenesis.

Endothelial cells line blood vessels, and their proliferation during neovascularization ( i.e., angiogenesis) is essential for a normal growth and development as well as for tumor progression and metastasis. Mechanistic details indicated that down-regulation of Glc(3)Man(9)GlcNAc(2)-PP-Dol level reduced angiogenesis and induced apoptosis in capillary endothelial cells (Martínez JA, Torres-Negrón I, Amigó LA, Banerjee DK, Cellular and Molec Biochem 45, 137-152 (1999)). Unlike in any other insulin-responsive cells, insulin reduced capillary endothelial cell proliferation by increasing the cell doubling time. But, when analyzed, the rate of lipid-linked oligosaccharide-PP-Dol (LLO) synthesis as well as its turnover ( i.e., t(1/2)) were increased in insulin treated cells. No major differences in their molecular size were observed. This corroborated with an enhanced glycosylation of Factor VIIIC, an N-linked glycoprotein (essential cofactor of the blood coagulation cascade) and a marker for the capillary endothelial cell. Increased LLO synthesis was independent of elevating either Dol-P level or Man-P-Dol synthase gene (dpm) transcription. Insulin however, enhanced 2-deoxy-glucose transport across the endothelial cell plasma membrane and caused increased secretion of Factor VIIIC, thus, supporting the existence of additional LLO pool(s), and arguing favorably that growth retardation of capillary endothelial cells by insulin turned a highly proliferative cell into a highly secretory cell.

Reversal of P-glycoprotein-mediated paclitaxel resistance by new synthetic isoprenoids in human bladder cancer cell line.

We isolated a paclitaxel-resistant cell line (KK47/TX30) from a human bladder cancer cell line (KK47/WT) in order to investigate the mechanism of and reversal agents for paclitaxel resistance. KK47/TX30 cells exhibited 700-fold resistance to paclitaxel and cross-resistance to vinca alkaloids and topoisomerase II inhibitors. Tubulin polymerization assay showed no significant difference in the ratio of polymerized alpha- and beta-tubulin between KK47/WT and KK47/TX30 cells. Western blot analysis demonstrated overexpression of P-glycoprotein (P-gp) and lung resistance-related protein (LRP) in KK47/TX30 cells. Drug accumulation and efflux studies showed that the decreased paclitaxel accumulation in KK47/TX30 cells was due to enhanced paclitaxel efflux. Cell survival assay revealed that verapamil and cepharanthine, conventional P-gp modulators, could completely overcome paclitaxel resistance. To investigate whether new synthetic isoprenoids could overcome paclitaxel resistance, we synthesized 31 isoprenoids based on the structure of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (SDB), which could reverse multidrug resistance (MDR), as shown previously. Among those examined, trans-N,N'-bis(3,4-dimethoxybenzyl)-N-solanesyl-1,2-diaminocyclohexane (N-5228) could completely reverse paclitaxel resistance in KK47/TX30 cells. N-5228 inhibited photoaffinity labeling of P-gp by [(3)H]azidopine, suggesting that N-5228 could bind to P-gp directly and could be a substrate of P-gp. Next, we investigated structural features of these 31 isoprenoids in order to determine the structural requirements for the reversal of P-gp-mediated paclitaxel resistance, suggesting that the following structural features are important for overcoming paclitaxel resistance: (1) a basic structure of 8 to 10 isoprene units, (2) a cyclohexane ring or benzene ring within the framework, (3) two cationic sites in close proximity to each other, and (4) a benzyl group with 3,4-dimethoxy functionalities, which have moderate electron-donating ability. These findings may provide valuable information for the development of P-gp-mediated MDR-reversing agents.

Stimulation by dolichol phosphate-mannose of N-acetylglucosaminyl-lipid biosynthesis by membranes from class E Thy-1-negative mutant mouse lymphoma cells which are defective in dolichol phosphate-mannose biosynthesis.

Dolichol phosphate-mannose has been shown previously to stimulate the biosynthesis of N-acetylglucosaminyl-diphosphate-dolichol (E. L. Kean (1985) J. Biol. Chem. 260, 12561-12571). Although the class E Thy-1-negative mutant mouse lymphoma cells are unable to synthesize dolichol phosphate-mannose, the addition of this compound exogenously to membranes from the mutant cells brought about a stimulation of N-acetylglucosaminyl-lipid synthesis similar to that obtained with membranes from wild type cells. The retention of this activity by the mutant cells supports the suggestion of a regulatory role for dolichol phosphate-mannose as an intrinsic property of the glucosaminyltransferase which catalyzes the initial reaction of the dolichol pathway.

Stimulation by dolichol phosphate-mannose and phospholipids of the biosynthesis of N-acetylglucosaminylpyrophosphoryl dolichol.

Dolichol phosphate-mannose (dol-P-mannose) has been shown previously to stimulate the reaction: dolichol phosphate + UDP-[3H]GlcNAc----[3H]GlcNAc-P-P-polyprenols (Kean, E. L. (1982) J. Biol. Chem. 257, 7952-7954). Further studies on this phenomenon are described, using microsomes from the retina of the embryonic chick as the major source of enzyme. Neither dolichol-P-glucose nor retinyl-P-mannose showed this stimulatory activity. Phosphatidylglycerol also stimulated this same process and was most active among a variety of phospholipids which were tested, in accord with previous reports. The presence of GDP-2-deoxy-2-fluoro-D-mannose or GTP had no effect on the reaction. The apparent activation constant for dolichol-P-mannose was 2.2 microM, and for phosphatidylglycerol, 401 microM. The major product (90% or greater) obtained under basal and stimulatory conditions was GlcNAc-P-P-dolichol and the site of the stimulatory effect was the glucosaminyltransferase catalyzing the formation of this compound. The effects of stimulation on the kinetic properties were similar for both activators: increases in the Vmax of the reactions of 7-10-fold; increases in apparent Km for UDP-GlcNAc of 5-7-fold; a 3-fold decrease in apparent Km for dolichol-phosphate. When present together, a mutual inhibition of stimulation was observed compared to the additive effect from dol-P-mannose or phosphatidylglycerol alone. Although a substrate for the reaction, dolichol phosphate repressed the stimulation by dolichol-P-mannose but not that by phosphatidylglycerol. Dol-P-glucose, while not an activator of the reaction, acted as a negative modifier of the stimulation by dol-P-mannose by acting as a competitive inhibitor of the stimulation. The stimulatory phenomenon was observed in microsomes prepared from a variety of tissues from the embryonic chick and from postnatal tissue after partial delipidation. The addition of pyrophosphatase inhibitors did not bring about stimulation of GlcNAc-lipid synthesis, but did enhance the effect. These studies extend the previous observations of the participation of dolichol-P-mannose and phosphatidylglycerol as allosteric activators of GlcNAc-lipid synthesis and indicate additional aspects of metabolic regulation of the dolichol pathway.