Cytoplasmic inositol hexakisphosphate production is sufficient for mediating the Gle1-mRNA export pathway

Production of inositol hexakisphosphate (IP6) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells. To examine the network of interactions that require IP6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors. Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function. Complementation of the nup116Deltaipk1Delta and nup42Deltaipk1Delta double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex. With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP6 may regulate a cytoplasmic step in mRNA export. To test this prediction, the spatial requirements for the production of IP6 were investigated. Restriction of Ipk1 to the cytoplasm did not block IP6 production. Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP6 production. Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm. Additionally, the cytoplasmic production of IP6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant. Thus, cytoplasmic IP6 production is sufficient for mediating the Gle1-mRNA export pathway.     

L-Canavanine as a radiosensitization agent for human pancreatic cancer cells

This study evaluated the in vitro effect of L-canavanine on cell cycle progression in the two human pancreatic cancer cells lines PANC-1 and MIA PaCa-2. After 72 h of exposure to L-canavanine, the percentage of cells in the radiosensitive G₂/M phase of the cell cycle increased 6-fold in PANC-1 cells and 4-fold in MIA PaCa-2 cells, when compared to untreated cells. The capacity of L-canavanine to redistribute cells into the G₂/M phase of the cell cycle was both concentration- and time-dependent. Since many drugs that cause cells to accumulate in the G₂/M phase of the cell cycle are effective radiosensitization agents, the potential of L-canavanine to synergistically enhance the effects of ionizing radiation also was evaluated. The interaction between these treatment modalities was quantified using the median-effect equation and combination index analysis. L-Canavanine was found to be synergistic with radiation when either PANC-1 or MIA PaCa-2 cells were exposed to L-canavanine for 72 h prior to irradiation. These results suggest that L-canavanine in combination with radiation may have clinical potential in the treatment of pancreatic cancer.     

The mechanism of L-canavanine cytotoxicity: arginyl tRNA synthetase as a novel target for anticancer drug discovery

There is a clear need for agents with novel mechanisms of action to provide new therapeutic approaches for the treatment of pancreatic cancer. Owing to its structural similarity to L-arginine, L-canavanine, the beta-oxa-analog of L-arginine, is a substrate for arginyl tRNA synthetase and is incorporated into nascent proteins in place of L-arginine. Although L-arginine and L-canavanine are structurally similar, the oxyguanidino group of L-canavanine is significantly less basic than the guanidino group of L-arginine. Consequently, L-canavanyl proteins lack the capacity to form crucial ionic interactions, resulting in altered protein structure and function, which leads to cellular death. Since L-canavanine is selectively sequestered by the pancreas, it may be especially useful as an adjuvant therapy in the treatment of pancreatic cancer. This novel mechanism of cytotoxicity forms the basis for the anticancer activity of L-canavanine and thus, arginyl tRNA synthetase may represent a novel target for the development of such therapeutic agents.     

Rab7 prevents growth factor-independent survival by inhibiting cell-autonomous nutrient transporter expression

Growth factor withdrawal results in the endocytosis and degradation of transporter proteins for glucose and amino acids. Here, we show that this process is under the active control of the small GTPase Rab7. In the presence of growth factor, Rab7 inhibition had no effect on nutrient transporter expression. In growth factor-deprived cells, however, blocking Rab7 function prevented the clearance of glucose and amino acid transporter proteins from the cell surface. When Rab7 was inhibited, growth factor deprived cells maintained their mitochondrial membrane potential and displayed prolonged, growth factor-independent, nutrient-dependent cell survival. Thus, Rab7 functions as a proapoptotic protein by limiting cell-autonomous nutrient uptake. Consistent with this, dominant-negative Rab7 cooperated with E1A to promote the transformation of p53(-/-) mouse embryonic fibroblasts (MEFs). These results suggest that proteins that limit nutrient transporter expression function to prevent cell-autonomous growth and survival.     

The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.     

The human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase

We have demonstrated that the human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase (InsP(4) 5-kinase). The cDNA of the human gene contained a putative open reading frame of 1251 bp encoding 416 amino acids with 83.6% identity compared with the rat protein. The substrate specificity of the recombinant human protein demonstrated preference for Ins(1,3,4,6)P(4) with a catalytic efficiency (V(max)/K(m)) 43-fold greater than that of Ins(1,3,4,5)P(4) and 2-fold greater than that of Ins(1,4,5)P(3). The apparent V(max) was 114 nmol of Ins(1,3,4,5,6)P(5) formed/min/mg of protein, and the apparent K(m) was 0.3 microm Ins(1,3,4,6)P(4). The functional homolog in yeast is Ipk2p, and ipk2-null yeast strains do not synthesize Ins(1,3,4,5,6)P(5) or InsP(6). Synthesis of these compounds was restored by transformation with wild-type yeast IPK2 but not with human InsP(4) 5-kinase. Thus the human gene does not complement for the loss of the yeast gene because yeast cells do not contain the substrate Ins(1,3,4,6)P(4), and the reaction of the human protein with Ins(1,3,4,5)P(4) is insufficient to effect rescue or synthesis of InsP(5) and InsP(6). Therefore the major activity of human InsP(4) 5-kinase is phosphorylation at the D-5 position, and the pathways for synthesis of Ins(1,3,4,5,6)P(5) in yeast versus humans are different.     

Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake

In multicellular organisms, constituent cells depend on extracellular signals for growth, proliferation, and survival. When cells are withdrawn from growth factors, they undergo apoptosis. Expression of constitutively active forms of the serine/threonine kinase Akt/PKB can prevent apoptosis upon growth factor withdrawal. Akt-mediated survival depends in part on the maintenance of glucose metabolism, suggesting that reduced glucose utilization contributes to growth factor withdrawal-induced death. However, it is unclear how restricting access to extracellular glucose alone would lead to the metabolic collapse observed after growth factor withdrawal. We report herein that growth factor withdrawal results in the loss of surface transporters for not only glucose but also amino acids, low-density lipoprotein, and iron. This coordinated decline in transporters and receptors for extracellular molecules creates a catabolic state characterized by atrophy and a decline in the mitochondrial membrane potential. Activated forms of Akt maintained these transporters on the cell surface in the absence of growth factor through an mTOR-dependent mechanism. The mTOR inhibitor rapamycin diminished Akt-mediated increases in cell size, mitochondrial membrane potential, and cell survival. These results suggest that growth factors control cellular growth and survival by regulating cellular access to extracellular nutrients in part by modulating the activity of Akt and mTOR.     

Early exposure to genistein exerts long-lasting effects on the endocrine and immune systems in rats

BACKGROUND: Although the immunologic effects of endogenous and synthetic estrogens are well studied, few studies have examined the hormonal effects of phytoestrogens (i.e., plant-derived estrogens) on the immune system. The primary goal of this study was to compare the effects of perinatal exposure with life-long exposure to genistein, an estrogenic compound in soy, on the endocrine and immune system in adulthood. MATERIALS AND METHODS: Pregnant female rats were exposed to no, low (5 mg/kg diet), or high (300 mg/kg diet) genistein diets throughout gestation and lactation. At weaning, male offspring exposed to genistein perinatally were either switched to the genistein-free diet or remained on the genistein-dosed diets. At 70 days of age, immune organ masses, lymphocyte subpopulations, cytokine concentrations, and testosterone concentrations were assessed in male offspring. RESULTS: Data were analyzed based on the diets that males were exposed to during gestation and lactation because life-long exposure to genistein had no additional effect on any of the dependent measures. Relative thymus masses were greater among males exposed to the high genistein diet than among males exposed to no genistein. Although the proportions of splenic and thymic CD4+ T cells were not altered by genistein, the percentages of CD4+CD8+ thymocytes, CD8+ splenocytes, and total T cells in the spleen were higher and the percentages of CD4-CD8- thymocytes were lower among males exposed to genistein than among males not exposed to genistein. Synthesis of interferon-gamma (IFN-gamma) was marginally higher and testosterone concentrations were lower among genistein-exposed than genistein-free males. DISCUSSION: These data illustrate that exposure to genistein during pregnancy and lactation exerts long-lasting effects on the endocrine and immune systems in adulthood. Whether exposure to phytoestrogens during early development affects responses to infectious or autoimmune diseases, as well as cancers, later in life requires investigation.