A New Mixed-Backbone Oligonucleotide against Glucosylceramide Synthase Sensitizes Multidrug-Resistant Tumors to Apoptosis

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C₁₈-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.     

Potentiation of cell killing by fractionated radiation and suppression of proliferative recovery in MCF-7 breast tumor cells by the Vitamin D3 analog EB 1089

A senescence-like growth arrest succeeded by recovery of proliferative capacity was observed in MCF-7 breast tumor cells exposed to fractionated radiation, 5 × 2 Gy. Exposure to EB 1089, an analog of the steroid hormone 1, 25 dihydroxycholecalciferol (1, 25 dihydroxy Vitamin D₃; calcitriol), prior to irradiation promoted cell death and delayed both the development of a senescent phenotype and the recovery of proliferative capacity. EB 1089 also reduced clonogenic survival over and above that produced by fractionated radiation alone and further conferred susceptibility to apoptosis in MCF-7 cells exposed to radiation. In contrast, EB 1089 failed to enhance the response to radiation (or to promote apoptosis) in normal breast epithelial cells or BJ fibroblast cells. EB 1089 treatment and fractionated radiation additively promoted ceramide generation and suppressed expression of polo-like kinase 1. Taken together, these data indicate that EB 1089 (and 1, 25 dihydroxycholecalciferol or its analogs) could selectively enhance breast tumor cell sensitivity to radiation through the promotion of cell death, in part through the generation of ceramide and the suppression of polo-like kinase.     

Corticosteroid-binding globulin synthesis and distribution in rat white adipose tissue

The lactone isolated from Fusarium termed L659,699 is a potent specific inhibitor of the enzyme 3hydroxi3methylglutaril coenzyme A (HMG-CoA) synthase. In cultures of smooth muscle cells (SMC) isolated from aortic-arch of control (CSMC) and 5% of cholesterol diet (Ch-SMC) treated chicks, the incorporation of (¹⁴C)acetate to lipids (cholesterol, triacylglycerides and cholesterol ester) were greater in ChSMC cultures than in CSMC and the presence of 0.05 M L659,699 for 2 h in the incubation medium decrease the synthesis of cholesterol however the triacylglycerides synthesis increase. The effect of inhibitor is stronger in young cultures (3–4 steps) than in the older ones (11–12 steps). In young CSMC and ChSMC cultures the inhibition of cholesterol and triacylglycerides synthesis by L659,699 was reversal.     

Ceramide signaling in fenretinide-induced endothelial cell apoptosis

Stress stimuli can mediate apoptosis by generation of the lipid second messenger, ceramide. Herein we investigate the molecular mechanism of ceramide signaling in endothelial apoptosis induced by fenretinide (N-(4-hydroxyphenyl)retinamide (4-HPR)). 4-HPR, a synthetic derivative of retinoic acid that induces ceramide in tumor cell lines, has been shown to have antiangiogenic effects, but the molecular mechanism of these is largely unknown. We report that 4-HPR was cytotoxic to endothelial cells (50% cytotoxicity at 2.4 microm, 90% at 5.36 microm) and induced a caspase-dependent endothelial apoptosis. 4-HPR (5 microm) increased ceramide levels in endothelial cells 5.3-fold, and the increase in ceramide was required to achieve the apoptotic effect of 4-HPR. The 4-HPR-induced increase in ceramide was suppressed by inhibitors of ceramide synthesis, fumonisin B(1), myriocin, and l-cycloserine, and 4-HPR transiently activated serine palmitoyltransferase, demonstrating that 4-HPR induced de novo ceramide synthesis. Sphingomyelin levels were not altered by 4-HPR, and desipramine had no effect on ceramide level, suggesting that sphingomyelinase did not contribute to the 4-HPR-induced ceramide increase. Finally, the pancaspase inhibitor, t-butyloxycarbonyl-aspartyl[O-methyl]-fluoromethyl ketone, suppressed 4-HPR-mediated apoptosis but not ceramide accumulation, suggesting that ceramide is upstream of caspases. Our results provide the first evidence that increased ceramide biosynthesis is required for 4-HPR-induced endothelial apoptosis and present a molecular mechanism for its antiangiogenic effects.     

Glucosylceramide synthase and apoptosis

Glucosylceramide synthase (GCS) is an enzyme inherent to ceramide metabolism. The enzyme catalyzes the transfer of glucose to ceramide, the first committed step in glycolipid biosynthesis. Known for many years as a branch point enzyme directing synthesis of cerebrosides and gangliosides, GCS has recently been implicated in the cytotoxic response of cancer cells to chemotherapy. With ceramide now occupying a central role in the signaling mechanisms of apoptosis, the position of GCS as sentry is perhaps not unexpected. In particular, it has been recognized that the toxic response of cells to chemotherapy is impaired when GCS activity is elevated and heightened when GCS activity is blocked. Herein we review the control points of ceramide metabolism with special regard to GCS and the cytotoxic response.     

Ion allocation in two different salt-tolerant Mediterranean Medicago species

The relationship between Na+, major cation concentrations and salt tolerance under long-term saline conditions of Medicago arborea and Medicago citrina was studied. Plants were grown in solution culture in 1, 50, 100, or 200 mmol/L NaCl for 30 days in a climate-controlled greenhouse. Stem and petiole growth was the most affected by salt in both species. Leaf growth was inhibited in M. arborea, with increased salt, while only the 200 mmol/L NaCl-treated M. citrina plants were significantly affected. Both species had the highest Na+ concentrations in the shoots, however, the allocation pattern was different; M. arborea showed the highest concentrations in the leaf blades, whereas M. citrina distributed the salt into the petioles. K+/Na+ ratio decreased with salt in both species; however, leaf K+ use efficiency (g leaf DW mg-1 leaf K+) was higher in M. citrina. The difference in Na+ allocation and cation concentrations found in these medic species and their importance is discussed in relation to their response to NaCl salinity.     

Vasopressin is the only component of serum-free medium that stimulates phosphatidylcholine hydrolysis and accumulation of diacylglycerol in cultured REF52 cells

Vasopressin stimulates phosphatidylcholine hydrolysis in REF52 cells, and this phosphatidylcholine hydrolysis results in increases in choline containing metabolites in the culture medium (2.3 x control levels) and accumulation of cellular diacylglycerol (6.5 x control levels). Vasopressin is the only component of a 6-component mixture of the serum-free medium for REF52 cells that induces the phosphatidylcholine hydrolysis response. The effect of vasopressin is both time- and concentration-dependent. Maximal levels of both phosphatidyl-choline hydrolysis and accumulation of diacylglycerol are observed between 10 and 20 min after treatment with vasopressin. Effects are maximal at vasopressin concentrations of 100 ng/ml; the ED50 for vasopressin-stimulated phosphatidyl-choline hydrolysis is approximately 0.7 ng/ml. The evolution of diacylglycerol occurs in a time frame that is consistent with the diacylglycerol activating protein kinase C in a "second phase" agonist response.     

Genetics of Reproductive Isolation in the Drosophila Simulans Clade: Complex Epistasis Underlying Hybrid Male Sterility

We have analyzed the sterility associated with introgressions of the distal one-fourth of the X chromosome from either Drosophila mauritiana or Drosophila sechellia into the genome of Drosophila simulans using a series of visible and DNA markers. Because in Drosophila hybrids, male sterility is usually complete and is often tightly linked with each of several markers used in crosses, a simple genetic basis has generally been assumed. In our low resolution mapping experiment, we were not able to reject the null hypothesis that a single gene, introgressed from either D. mauritiana or D. sechellia, is the cause of male sterility. High resolution mapping, however, reveals a much more complex picture. At least three distinct factors from D. mauritiana, or two from D. sechellia, were identified that need to be jointly present to confer full sterility. Each individual factor by itself is relatively ineffective in causing sterility, or even a partial spermatogenic defect. Moreover, there appear to be more sterility factors on comparable introgressions from D. mauritiana than from D. sechellia. On the basis of these observations, we propose a model which suggests that multilocus weak allele interactions are a very common cause of reproductive incompatibility between closely related species. We also present theoretical argument and empirical evidence against extrapolating the results of within-species analysis to interpret the genetic basis of species differences. The implications of this model on the theories of evolution of species differences and the attempt to understand the mechanisms of hybrid sterility/inviability at the molecular level are discussed.