Elevated calcium in preneoplastic cells activates NF-kappa B and confers resistance to apoptosis.

Early preneoplastic cells (sup+) exhibit increased susceptibility to apoptosis, which is lost in late stage preneoplastic cells (sup-). Sup+ cells, which undergo apoptosis when cultured in low serum, show little or no DNA binding activity to nuclear factor (NF)-kappa B either in 10% or 0.2% serum. In contrast sup- cells, which are resistant to apoptosis in low serum, show a sustained constitutive activation of NF-kappa B. The constitutive activation of NF-kappa B observed in sup- cells is not due to loss of I kappa B alpha. We considered that the activation of NF-kappa B in sup- cells might be secondary to an increase in cytosolic Ca(2+), since sup- cells have a cytosolic Ca(2+) level that is double that in sup+ cells. In support of a role for Ca(2+), lowering cytosolic Ca(2+) in sup- cells by addition of the cell-permeable Ca(2+) chelator 1,2 bis(O-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) reduced cytosolic Ca(2+) by approximately 31% relative to untreated sup- cells, concomitant with a 65% reduction in NF-kappa B DNA binding activity and a reduction in I kappa B kinase (IKK) activity. In sup- cells in low serum, addition of BAPTA-AM also resulted in a significant ( approximately 50%) increase in caspase-3 activity. Raising extracellular Ca(2+) in sup+ cells resulted in a slight activation of I kappa B kinase and in enhanced NF-kappa B DNA binding activity. Using proteasome and calpain inhibitors, we determined that the basal activity of NF-kappa B in sup- cells is largely proteasome-independent, but sensitive to calpain inhibitors. Taken together these data suggest that the elevated Ca(2+) in sup- cells causes a modest activation of IKK, which likely contributes to the enhanced basal activation of NF-kappa B in sup- cells; however, the predominant effect of Ca(2+) appears to be mediated by Ca(2+)-enhanced degradation by calpain.

The oncostatic action of melatonin in an ovarian carcinoma cell line.

Melatonin is reported to reduce proliferation in many cell types, but the effect is small and the results are inconsistent. Information on the mechanism by which melatonin exerts its antiproliferative effects might provide insight into the variability of the response. In an ovarian adenocarcinoma cell line (BG-1), we find that melatonin at concentrations of 10(-9)-10(-7) M caused a 20-25% reduction in cell number. Melatonin also resulted in a similar reduction in [3H]-thymidine incorporation with no significant increase in cell death as measured by trypan blue incorporation. The Kd for melatonin reduction in cell number was approximately 5 x 10(-10) M. Melatonin ML2 receptors have a Kd for melatonin binding in the low nM range and are linked to the production of the calcium mobilizing agent inositol-1,4,5-trisphosphate (IP3). To investigate whether melatonin signaling involves an increase in cytosolic-free calcium. BG-1 cells were loaded with the calcium sensitive indicator, fura-2. Acute addition of melatonin (10(-5)-10(-9) M) did not alter cytosolic calcium. Addition of the putative nuclear receptor agonist CGP52608 caused a dose-dependent inhibition of cell number with a Kd of approximately 2 x 10(-9) M. Addition of CGP52608 caused a similar reduction in [3H]-thymidine incorporation. Neither melatonin (10(-8) M-10(-5) M) nor CGP52608 at concentrations below 10(-7) M induced cell death associated with the inhibition of cell proliferation; however, addition of CGP52608 at a high dose (10(-7) M) caused an increase in cell death, consistent with apoptosis. Growth inhibition by melatonin or CGP52608 did not alter the percentage of cells in G1 versus S/G2/M.

Reduced capacitative calcium entry correlates with vesicle accumulation and apoptosis.

A preneoplastic variant of Syrian hamster embryo cells, sup(+), exhibits decreased endoplasmic reticulum calcium levels and subsequently undergoes apoptosis in low serum conditions (Preston, G. A., Barrett, J. C., Biermann, J. A., and Murphy, E. (1997) Cancer Res. 57, 537-542). This decrease in endoplasmic reticulum calcium appears to be due, at least in part, to reduced capacitative calcium entry at the plasma membrane. Thus we investigated whether inhibition of capacitative calcium entry per se could reduce endoplasmic reticulum calcium and induce apoptosis of cells. We find that treatment with either SKF96365 (30-100 microM) or cell-impermeant 1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N', N'-tetraacetic acid (5-10 mM) is able to induce apoptosis of cells in conditions where apoptosis does not normally occur. Because previous work has implicated vesicular trafficking as a mechanism of regulating capacitative calcium entry, we investigated whether disruption of vesicular trafficking could lead to decreased capacitative calcium entry and subsequent apoptosis of cells. Coincident with low serum-induced apoptosis, we observed an accumulation of vesicles within the cell, suggesting deregulated vesicle trafficking. Treatment of cells with bafilomycin (30-100 nM), an inhibitor of the endosomal proton ATPase, produced an accumulation of vesicles, decreased capacitative entry, and induced apoptosis. These data suggest that deregulation of vesicular transport results in reduced capacitative calcium entry which in turn results in apoptosis.

Yeast N1e3p/Nup170p is required for normal stoichiometry of FG nucleoporins within the nuclear pore complex.

The FG nucleoporins are a conserved family of proteins, some of which bind to the nuclear localization sequence receptor, karyopherin. Distinct members of this family are found in each region of the nuclear pore complex (NPC), spanning from the cytoplasmically disposed filaments to the distal end of the nuclear basket. Movement of karyopherin from one FG nucleoporin to the next may be required for translocation of substrates across the NPC. So far, nothing is known about how the FG nucleoporins are localized within the NPC. To identify proteins that interact functionally with one member of this family, the Saccharomyces cerevisiae protein Nup1p, we previously identified 16 complementation groups containing mutants that are lethal in the absence of NUP1 These mutants were referred to as nle (Nup-lethal) mutants. Mutants in the nle3/nlel7 complementation group are lethal in combination with amino-terminal nup1 truncation mutants, which we have previously shown to be defective for localization to the NPC. Here we show that NLE3 (which is allelic to NUP170) encodes a protein with similarity to the mammalian nucleoporin Nup155. We show that Nle3p coprecipitates with glutathione S-transferase fusions containing the amino-terminal domain of Nup1p. Furthermore, a deletion of Nle3p leads to changes in the stoichiometry of several of the XFXFG nucleoporins, including the loss of Nup1p and Nup2p. These results suggest that Nle3p plays a role in localizing specific FG nucleoporins within the NPC. The broad spectrum of synthetic phenotypes observed with the nle3delta mutant provides support for this model. We also identify a redundant yeast homolog that can partially substitute for Nle3p and show that together these proteins are required for viability.

Structure-function relationships of the complement regulatory protein, CD59.

CD59 (membrane inhibitor of reactive lysis, protectin) is a membrane protein whose functions include the inhibition of the insertion of the ninth component of complement into the target membrane. It belongs to a superfamily of proteins including Ly-6, elapid snake venom toxins, and urokinase receptor (UPAR); the members of the superfamily have a similar structure that includes four (in mammals five) disulfide bridges that maintain a three-dimensional conformation consisting of a central core, three finger-like "loops" extending from it and a small loop near the coboxyl end. We have used site directed mutagenesis to explore three aspects of the structure of CD59: 1) the role of the disulfide bridges in expression and function of the molecule; 2) the location of epitopes reacting with monoclonal antibodies to the molecule; and 3) the parts of the molecule that are critical to its function in inhibiting complement lysis. Mutant molecules in which the disulfides maintaining the finger-like loops (Cys3-Cys26, Cys19-Cys39, and Cys45-Cys63) were removed were not expressed on the cell surface. The mutation of the disulfide (Cys6-Cys13) resulted in no change in expression or function. The mutation of Cys64-Cys69 maintaining the small loop resulted in an expressed molecule with increased functional activity. The major epitope for 6 of 7 monoclonal antibodies was centered on Arg53 as the mutation 53Arg-->Ser resulted in a loss of interaction with these antibodies, as did the deletion of four nearby residues (Leu54-Asn57). The alteration 55Arg-->Ser resulted in loss of reactivity for some but not other antibodies. The reactivity with one monoclonal antibody, H19, was abrogated by the mutations 61Tyr-->Gly and 61Tyr-->Ala. Functional activity of the molecule was not adversely altered by mutations in the first and second loops; however, the 61Tyr-->Gly mutation was non-functional. The mutation of 61Tyr-->His diminished function but changes 61Tyr-->Ala and 61Tyr-->Phe had no effect on function. We conclude that the functional site of CD59 is located in this region of the molecule.

The structure of the urokinase-type plasminogen activator receptor gene.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that plays a central role in pericellular plasminogen activation. It contains 313 amino acid residues, including 28 cysteine residues in a pattern of three homologous repeats. The cysteine residue pattern suggests that uPAR belongs to a superfamily of proteins including CD59, murine Ly-6, and a variety of elapid snake venom toxins. A novel 1.7-kb uPAR cDNA was isolated that is missing exon 5 and that contains 380 bp not previously reported at the 5' end. This cDNA was used to probe a human genomic library from which three clones were isolated and analyzed. The uPAR gene consists of 7 exons spread over 23 kb of genomic DNA. Exons 2, 4, and 6 code for homologous domains within the mature protein, as do exons 3, 5, and 7; CD59-like homologous pairs are encoded by exons 2-3, 4-5, and 6-7, respectively. The structure of the gene for uPAR further confirms the relationship of this molecule to the superfamily containing CD59, Ly-6, and the elapid snake venom toxins.

Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein.

The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function.

Stream drift, size-specific predation, and the evolution of ovum size in an amphibian.

A stream-breeding race of small-mouthed salamanders (Ambystoma texanum) in central Kentucky produces ova that are twice as large as those of a pond-breeding race found nearby. Embryos of stream-breeders also hatch at a more advanced developmental stage than those of pond-breeders. Morphological evidence indicates that stream-breeders were derived from pond-breeding stock. Assuming that differences between stream and pond-breeders reflect evolutionary change, and that the ancestral pond stock that invaded streams was similar to extant pond-breeders, we examined three hypotheses that might explain changes in ovum size and stage at hatching following the invasion of streams. (1) Larger ovum size evolved indirectly as a consequence of selection for rapid development which minimizes mortality risk from stream drying. (2) Increased ovum (hatchling) size and stage at hatching of stream-breeders are adaptations to resist stream current. (3) Increased ovum (hatchling) size and stage at hatching are adaptations to reduce predation on hatchlings from stream invertebrates. The results of field and laboratory studies only support hypotheses (2) and (3). Hatchlings that were relatively large or at a more advanced developmental stage had slower drift rates and were less vulnerable to predation by Phagocata gracilis, a flatworm abundant in streams in central Kentucky. Developmental and growth parameters were not correlated significantly with ovum size in populations of either geographic race. Differences in degree of parental care among races also cannot explain variation in ovum size since both races abandon their eggs immediately after oviposition.

Prey size-distributions and size-specific foraging success of Ambystoma larvae.

We examined how prey size-distributions influence size-specific foraing rate and food gain, i.e., food intake scaled to metabolic demands, in Jefferson's and small-mouth salamander larvae. Ambystoma jeffersonianum larvae sampled on 17 dates from a farm pond whose fauna was dominated by macrozooplankton and chironomid larvae were rarely gape-limited, and total volume of food in the stomach (VS) showed only a slight tendency to increase with larval size. Although 15 of 17 correlation coefficients of VS with larval size were positive, only 1 of 17 correlations were statistically significant, and body size explained only 8% of the overall variation in VS. Correlation coefficients of food gain and body size were positive in 9 cases and negative in 8, but only 3 were statistically significant.In contrast, Ambystoma texanum larvae in 42 samples taken from five sites dominated by macrozooplankton as well as relatively large isopods and amphipods were almost always gape-limited, and VS tended to increase markedly with larval size. 40 of 42 correlation coefficients of VS and larval size were positive, and 19 correlations were statistically significant. Body size in turn explained about 35% of the overall variation in VS. Correlation coefficients of food gain and larval size were positive in 32 of 42 samples, and 9 of 10 significant correlations were positive.When food is limiting and prey selection is not limited by gape, smaller larvae may grow as fast or in some cases faster than larger larvae because they are nearly as effective foragers, but have lower metabolic demands. Larger larvae may in turn grow faster than smaller larvae in environments which support a broad size spectrum of prey, particularly when gape limitations are highly disproportionate among size classes. The growth rate of larvae in one size class relative to another depends primarily on the extent to which increased foraging rate compensates for higher energy demands as body size increases. Size-specific foraging rate may in turn be strongly influenced by the prey size-distribution within a habitat. These relationships suggest that relative size is not always a good a priori predictor of exploitative competitive ability.

Hatching of amphibian embryos: the physiological trigger.

Marbled salamander embryos hatch in water if the environmental oxygen pressure is 86 torr or less, but do not hatch if the environmental oxygen pressure is equivalent to that of air. Under hypoxic conditions, embryos hatch in aqueous and nonaqueous media with equal success. Increasing carbon dioxide pressure does not induce hatching, but does decrease the time to hatching by altering environmental pH.