Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA.

We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers.

A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.

Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse.

This study examines the question of whether apolipoprotein E (apoE) alters steady-state concentrations of plasma cholesterol carried in low density lipoproteins (LDL-C) by acting as a competitive inhibitor of hepatic LDL uptake or by altering the rate of net cholesterol delivery from the intestinal lumen to the liver. To differentiate between these two possibilities, rates of cholesterol absorption and synthesis and the kinetics of hepatic LDL-C transport were measured in vivo in mice with either normal (apoE+/+) or zero (apoE-/-) levels of circulating apoE. Rates of cholesterol absorption were essentially identical in both genotypes and equaled approximately 44% of the daily dietary load of cholesterol. This finding was consistent with the further observation that the rates of cholesterol synthesis in the liver (approximately 2,000 nmol/h) and extrahepatic tissues (approximately 3,000 nmol/h) were also essentially identical in the two groups of mice. However, the apparent Michaelis constant for receptor-dependent hepatic LDL-C uptake was markedly lower in the apoE-/- mice (44 +/- 4 mg/dl) than in the apoE+/+ animals (329 +/- 77 mg/dl) even though the maximal transport velocity for this uptake process was essentially the same (approximately 400 micrograms/h per g) in the two groups of mice. These studies, therefore, demonstrate that apoE-containing lipoproteins can act as potent competitive inhibitors of hepatic LDL-C transport and so can significantly increase steady-state plasma LDL-C levels. This apolipoprotein plays no role, however, in the regulation of cholesterol absorption, sterol biosynthesis, or hepatic LDL receptor number, at least in the mouse.

Basolateral membrane Na+/H+ exchange enhances HCO3- absorption in rat medullary thick ascending limb: evidence for functional coupling between basolateral and apical membrane Na+/H+ exchangers.

The role of basolateral membrane Na+/H+ exchange in transepithelial HCO3- absorption (JHCO3) was examined in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. In Na(+)-free solutions, addition of Na+ to the bath resulted in a rapid, amiloride-sensitive increase in intracellular pH. In MTALs perfused and bathed with solutions containing 146 mM Na+ and 25 mM HCO3-, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 microM) reversibly inhibited JHCO3 by 50%. Evidence that the inhibition of JHCO3 by bath amiloride was the result of inhibition of Na+/H+ exchange included the following: (i) the IC50 for amiloride was 5-10 microM, (ii) EIPA was a 50-fold more potent inhibitor than amiloride, (iii) the inhibition by bath amiloride was Na+ dependent, and (iv) significant inhibition was observed with EIPA as low as 0.1 microM. Fifty micromolar amiloride or 1 microM EIPA inhibited JHCO3 by 35% when added to the bath but had no effect when added to the tubule lumen, indicating that addition of amiloride to the bath did not directly inhibit apical membrane Na+/H+ exchange. In experiments in which apical Na+/H+ exchange was assessed from the initial rate of cell acidification following luminal EIPA addition, bath EIPA secondarily inhibited apical Na+/H+ exchange activity by 46%. These results demonstrate basolateral membrane Na+/H+ exchange enhances transepithelial HCO3- absorption in the MTAL. This effect appears to be the result of cross-talk in which an increase in basolateral membrane Na+/H+ exchange activity secondarily increases apical membrane Na+/H+ exchange activity.

An apical permeability barrier to NH3/NH4+ in isolated, perfused colonic crypts.

Fermentation of nonabsorbed nutrients in the colon generates high concentrations of NH3/NH4+ in the colonic lumen. NH3 is a small, lipophilic neutral weak base that readily permeates almost all cell membranes, whereas its conjugate weak acid NH4+ generally crosses membranes much more slowly. It is not known how colonocytes maintain intracellular pH in the unusual acid-base environment of the colon, where permeant acid-base products of fermentation exist in high concentration. To address this issue, we hand dissected and perfused single, isolated crypts from rabbit proximal colon, adapting techniques from renal-tubule microperfusion. Crypt perfusion permits control of solutions at the apical (luminal) and basolateral (serosal) surfaces of crypt cells. We assessed apical- vs. basolateral-membrane transport of NH3/NH4+ by using fluorescent dyes and digital imaging to monitor intracellular pH of microvacuolated crypt cells as well as luminal pH. We found that, although the basolateral membranes have normal NH3/NH4+ permeability properties, there is no evidence for transport of either NH3 or NH4+ across the apical borders of these crypt cells. Disaggregating luminal mucus did not increase the transport of NH3/NH4+ across the apical border. We conclude that, compared to the basolateral membrane, the apical border of crypt colonocytes has a very low permeability-area product for NH3/NH4+. This barrier may represent an important adaptation for the survival of crypt cells in the environment of the colon.

BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum.

Two interacting heat shock cognate proteins in the lumen of the yeast endoplasmic reticulum (ER), Sec63p and BiP (Kar2p), are required for posttranslational translocation of yeast alpha-factor precursor in vitro. To investigate the role of these proteins in cotranslational translocation, we examined the import of invertase into wild-type, sec63, and kar2 mutant yeast membranes. We found that Sec63p and Kar2p are necessary for both co- and posttranslational translocation in yeast. Several kar2 mutants, one of which had normal ATPase activity, were defective in cotranslational translocation of invertase. We conclude that the requirement for BiP/Kar2p, which is not seen in a reaction reconstituted with pure mammalian membrane proteins [Görlich, D. & Rapoport, T.A. (1993) Cell 75, 615-630], is not due to a distinction between cotranslational translocation in mammalian cells and posttranslational translocation in yeast cells.

Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach.

We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1-kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach. PsbW, the gene encoding this polypeptide, is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues which directs the N terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide does not require a delta pH across the membrane, nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously in an as yet unknown way. The PsbW mRNA levels are light regulated. Similar to cytochrome b559 and PsbS, but different from the chlorophyll-complexing polypeptides D1, D2, CP43, and CP47 of photosystem II, PsbW is present in etiolated spinach seedlings.

Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization.

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial mitogen that is able to induce angiogenesis in vivo [Leung, D. W., Cachianes, G., Kuang, W.-J., Goeddel, D. V. & Ferrara, N. (1989) Science 246 1306-1309]. To determine if VEGF also influences the behavior of primordial endothelial cells, we used an in vivo vascular assay based on the de novo formation of vessels. Japanese quail embryos injected with nanomolar quantities of the 165-residue form of VEGF at the onset of vasculogenesis exhibited profoundly altered vessel development. In fact, the overall patterning of the vascular network was abnormal in all VEGF-injected embryos. The malformations were attributable to two specific endothelial cell activities: (i) inappropriate neovascularization in normally avascular areas and (ii) the unregulated, excessive fusion of vessels. In the first instance, supernumerary vessels directly linked the inflow channel of the heart to the aortic outflow channel. The second aberrant activity led to the formation of vessels with abnormally large lumens. Ultimately, unregulated vessel fusion generated massive vascular sacs that obliterated the identity of individual vessels. These observations show that exogenous VEGF has an impact on the behavior of primordial endothelial cells engaged in vasculogenesis, and they strongly suggest that endogenous VEGF is important in vascular patterning and regulation of vessel size (lumen formation).

The bacterial colicin active against tumor cells in vitro and in vivo is verotoxin 1.

We have identified verotoxin 1 (VT1) as the active component within an antineoplastic bacteriocin preparation from Escherichia coli HSC10 studied over two decades. Recombinant VT1 can simulate the toxicity of anticancer proteins (ACP), and the antineoplastic activity of ACP (and VT1) was abrogated by treatment with anti-VT1 antibody. Similarly, VT1 mimics the protective effect of ACP in a murine metastatic fibrosarcoma model. Prior immunization with VT1 B subunit prevents the effect of VT1 or ACP in this model. The activity of ACP against a variety of human ovarian cell lines was mimicked by VT1, and multidrug-resistant variants were significantly hypersensitive. Primary ovarian tumors and metastases contain elevated levels of globotriaosylceramide compared with normal ovaries, and overlay of frozen tumor sections showed selective VT binding to tumor tissue and the lumen of invading blood vessels. Our contention that VT1 could provide an additional approach to the management of certain human neoplasms is discussed.

Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids.

It has been suggested that transepithelial gradients of short-chain fatty acids (SCFAs; the major anions in the colonic lumen) generate pH gradients across the colonic epithelium. Quantitative confocal microscopy was used to study extracellular pH in mouse distal colon with intact epithelial architecture, by superfusing tissue with carboxy SNARF-1 (a pH-sensitive fluorescent dye). Results demonstrate extracellular pH regulation in two separate microdomains surrounding colonic crypts: the crypt lumen and the subepithelial tissue adjacent to crypt colonocytes. Apical superfusion with (i) a poorly metabolized SCFA (isobutyrate), (ii) an avidly metabolized SCFA (n-butyrate), or (iii) a physiologic mixture of acetate/propionate/n-butyrate produced similar results: alkalinization of the crypt lumen and acidification of subepithelial tissue. Effects were (i) dependent on the presence and orientation of a transepithelial SCFA gradient, (ii) not observed with gluconate substitution, and (iii) required activation of sustained vectorial acid/base transport by SCFAs. Results suggest that the crypt lumen functions as a pH microdomain due to slow mixing with bulk superfusates and that crypts contribute significant buffering capacity to the lumen. In conclusion, physiologic SCFA gradients cause polarized extracellular pH regulation because epithelial architecture and vectorial transport synergize to establish regulated microenvironments.

Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts.

The predominant localization of the major auxin-binding protein (ABP1) of maize is within the lumen of the endoplasmic reticulum. Nevertheless, all the electrophysiological evidence supporting a receptor role for ABP1 implies that a functionally important fraction of the protein must reside at the outer face of the plasma membrane. Using methods of protoplast preparation designed to minimize proteolysis, we report the detection of ABP at the surface of maize coleoptile protoplasts by the technique of silver-enhanced immunogold viewed by epipolarization microscopy. We also show that ABP clusters following auxin treatment and that this response is temperature-dependent and auxin-specific.