Effect of appendicectomy on colonic inflammation and neoplasia in experimental ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) promotes cancer, and can be ameliorated by early appendicectomy for appendicitis. The aim of the study was to explore the effect of appendicectomy on colitis and colonic neoplasia in an animal model of colitis and a cohort of patients with UC. METHODS: Five-week old IL10/Nox1(DKO) mice with nascent colitis and 8-week-old IL10/Nox1(DKO) mice with established colitis underwent appendicectomy (for experimental appendicitis or no appendicitis) or sham laparotomy. The severity and extent of colitis was assessed by histopathological examination, and a clinical disease activity score was given. From a cohort of consecutive patients with UC who underwent colectomy, the prevalence of appendicectomy and pathological findings were collected from two institutional databases. RESULTS: Appendicectomy for appendicitis ameliorated experimental colitis in the mice; the effect was more pronounced in the 5-week-old animals. Appendicectomy in the no-appendicitis group was associated with an increased rate of colonic high-grade dysplasia (HGD) or cancer compared with rates in sham and appendicitis groups (13 of 20 versus 0 of 20 and 0 of 20 respectively; P < 0·001). Fifteen of 232 patients who underwent colectomy for UC had previously had an appendicectomy, and nine of these had colonic cancer or HGD. Thirty (13·8 per cent) of 217 patients with the appendix in situ had colonic neoplastic lesions. Multivariable analysis showed that previous appendicectomy was associated with colorectal neoplasia (odds ratio 16·88, 95 per cent c.i. 3·32 to 112·69). CONCLUSION: Appendicectomy for experimental appendicitis ameliorated colitis. The risk of colorectal neoplasia appeared to increase following appendicectomy without induced appendicitis in a mouse model of colitis, and in patients with UC who had undergone appendicectomy. Surgical relevance Appendicectomy for appendicitis protects against UC. In this murine model of colitis, appendicectomy for experimental appendicitis protected against colitis, but appendicectomy without appendicitis promoted colorectal carcinogenesis. In patients with ulcerative colitis who underwent colectomy, absence of the appendix (proof of previous appendicectomy) in the resection specimen was independently associated with colorectal neoplasia. Although patients with UC and a history of appendicectomy represent a small subset, they may need closer monitoring for colorectal neoplasia.

Ectopic expression of OX1R in ulcerative colitis mediates anti-inflammatory effect of orexin-A.

Orexins (orexin-A and orexin-B) are hypothalamic peptides that are produced by the same precursor and are involved in sleep/wake control, which is mediated by two G protein-coupled receptor subtypes, OX1R and OX2R. Ulcerative colitis (UC) is an inflammatory bowel disease, (IBD) which is characterized by long-lasting inflammation and ulcers that affect the colon and rectum mucosa and is known to be a significant risk factor for colon cancer development. Based on our recent studies showing that OX1R is aberrantly expressed in colon cancer, we wondered whether orexin-A could play a role in UC. Immunohistochemistry studies revealed that OX1R is highly expressed in the affected colonic epithelium of most UC patients, but not in the non-affected colonic mucosa. Injection of exogenous orexin-A specifically improved the inflammatory symptoms in the two colitis murine models. Conversely, injection of inactive orexin-A analog, OxB7-28 or OX1R specific antagonist SB-408124 did not have anti-inflammatory effect. Moreover, treatment with orexin-A in DSS-colitis induced OX1R-/- knockout mice did not have any protective effect. The orexin-A anti-inflammatory effect was due to the decreased expression of pro-inflammatory cytokines in immune cells and specifically in T-cells isolated from colonic mucosa. Moreover, orexin-A inhibited canonical NFκB activation in an immune cell line and in intestinal epithelial cell line. These results suggest that orexin-A might represent a promising alternative to current UC therapies.

Increased UDP-GlcNAc: alpha-mannoside beta(1----4) N-acetylglucosaminyltransferase activity during chick embryo development.

In vitro assays for the beta-N-acetylglucosaminyltransferases (GlcNAcTase) were performed on crude microsomal fractions prepared from 8-day chick embryo fibroblasts (8-day-CEF) and 16-day chick embryo fibroblasts (16-day-CEF) using [3H]mannose-labeled GlcNAc beta 1----2 Man alpha 1----6 (GlcNAc beta 1----2 Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (Fuc alpha 1----6) GlcNAc-Asn and UDP-GlcNAc as substrates. 8-day-CEF synthesize preferentially triantennary complex type chains, whereas 16-day-CEF produce essentially tetraantennary complex type chains. Furthermore oligosaccharides containing the GlcNAc beta 1----4 Man alpha 1----3 Man sequence represent 90% of the structures found in 16-day-CEF versus 30% in 8-day-CEF, indicating an increase in beta-N-acetylglucosaminyltransferase IV activity during embryo development.

Alteration of mannose transport in fibroblasts from type I carbohydrate deficient glycoprotein syndrome patients.

The aim of the present study was to explore how mannose enters fibroblasts derived from a panel of children suffering from different subtypes of type I carbohydrate deficient glycoprotein syndrome: seven carbohydrate deficient glycoprotein syndrome subtype Ia (phosphomannomutase deficiency), two carbohydrate deficient glycoprotein syndrome subtype Ib (phosphomannose isomerase deficiency) and two carbohydrate deficient glycoprotein syndrome subtype Ix (not identified deficiency). We showed that a specific mannose transport system exists in all the cells tested but has different characteristics with respect to carbohydrate deficient glycoprotein syndrome subtypes. Subtype Ia fibroblasts presented a mannose uptake equivalent or higher (maximum 1.6-fold) than control cells with a D-[2-3H]-mannose incorporation in nascent N-glycoproteins decreased up to 7-fold. Compared to control cells, the mannose uptake was greatly stimulated in subtype Ib (4.0-fold), due to lower Kuptake and higher Vmax values. Subtype Ib cells showed an increased incorporation of D-[2-3H]-mannose into nascent N-glycoproteins. Subtype Ix fibroblasts presented an intermediary status with mannose uptake equivalent to the control but with an increased incorporation of D-[2-3H]-mannose in nascent N-glycoproteins. All together, our results demonstrate quantitative and/or qualitative modifications in mannose transport of all carbohydrate deficient glycoprotein syndrome fibroblasts in comparison to control cells, with a relative homogeneity within a considered subtype of carbohydrate deficient glycoprotein syndrome. These results are consistent with the possible use of mannose as a therapeutic agent in carbohydrate deficient glycoprotein syndrome Ib and Ix.

Neuronal expression of the NADPH oxidase NOX4, and its regulation in mouse experimental brain ischemia.

Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia. We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry. Ischemia, induced by middle cerebral artery occlusion resulted in a dramatic increase in cortical NOX4 expression. Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15. The early onset suggests neuronal reaction, while the peak period corresponds to the time of neoangiogenesis occurring mainly in the peri-infarct region. The occurrence of NOX4 in the new capillaries was confirmed by immunohistochemical staining. In summary, our paper reports the presence of the ROS producing NADPH oxidase NOX4 in neurons and demonstrates an upregulation of its expression under ischemic conditions. Moreover, a role for NOX4 in ischemia/hypoxia-induced angiogenesis is suggested by its prominent expression in newly formed capillaries.

Signal transduction pathways in macroautophagy.

Macroautophagy is a major cellular catabolic pathway involved in the regulation of cell homeostasis. It is initiated by the sequestration of intracellular material by a wrapping membrane and terminates with the fusion of autophagic vacuoles with the lysosomal compartment. Macroautophagy has been extensively studied at the morphological level and in terms of environmental responses (nutrient deprivation, hormones). Recently a burst of data has emerged concerning the intracellular molecular events involved in the control of macroautophagic sequestration. It is becoming clear that the initial sequestration step of macroautophagy is under the control of different signalling pathways.

Relationship between the content of [14C]glucose-derived monosaccharides in glycoprotein oligosaccharide chains and the state of enterocytic differentiation of HT-29 cells.

The HT-29 cell line derived from a human colon adenocarcinoma has a glucose-dependent state of differentiation which is negatively correlated with the presence of D-glucose in the culture medium. The contribution of glucose to the biosynthesis of N-glycan chains, as a function of the differentiation state of HT-29 cells, was shown by: (a) [14C]glucose incorporation by undifferentiated HT-29 cells being lower after 2 h and higher after 19 h of metabolic labeling than that by differentiated cells; (b) a lack of glucose in the culture medium of undifferentiated HT-29 cells diminishing [14C]glucose incorporation into glycan chains, but not changing the glucose distribution between lipid- and protein-linked saccharides; (c) glucose behavior in undifferentiated HT-29 cells being not related to mannose-glycan metabolism, as the high-mannose compounds labeled with glucose and observed by HPLC showing a different distribution associated with the duration of glucose labeling; and (d) glucose being interconverted into other monosaccharide-glycan constituents in proportions different in differentiated and undifferentiated cell populations.

Heat shock protein gp96 and NAD(P)H oxidase 4 play key roles in Toll-like receptor 4-activated apoptosis during renal ischemia/reperfusion injury.

Ischemia/reperfusion injury (IRI) causes inflammation and cell injury as a result of activating innate immune signaling. Toll-like receptor 4 (TLR4) has a key role in mediating kidney damages during IRI, but the downstream signaling pathway(s) stimulating apoptosis remains debated. In this study we show that TLR4 mediates MyD88-dependent activation of TNF receptor-associated factor 2, apoptosis signal-regulating kinase 1 (ASK1), and Jun N-terminal kinase (JNK) and p38 MAP kinases in ischemic-reperfused kidneys and posthypoxic renal tubule epithelial cells (RTECs). Hypoxia stimulated the expression of the endoplasmic-resident gp96, which co-immunoprecipitated TLR4, whereas silencing gp96 mRNA expression impaired hypoxia-induced apoptosis in TLR4-expressing RTECs. NAD(P)H oxidase 4 (NOX4) was shown to interact with TLR4 and to be required in lipopolysaccharide-induced production of reactive oxygen species (ROS). IRI stimulated the expression of a 28-kDa NOX4 spliced isoform abundantly expressed in wild-type RTECs, which co-immunoprecipitated with TLR4, but not with gp96 in TLR4-deficient RTECs. Silencing NOX4 mRNA expression impaired hypoxia-induced activation of ASK1 and both JNK and p38, leading to the inhibition of ROS production and apoptosis in posthypoxic TLR4-expressing RTECs. These findings show that, concomitantly to the activation of p38, the gp96/TLR4 interaction is required for activation of ASK1/JNK signaling in posthypoxic mouse RTECs, and that the 28-kDa NOX4 has a key role in TLR4-mediated apoptosis during renal IRI.

The processing of asparagine-linked oligosaccharides in HT-29 cells is a function of their state of enterocytic differentiation. An accumulation of Man9,8-GlcNAc2-Asn species is indicative of an impaired N-glycan trimming in undifferentiated cells.

Studies on the regulation of the enterocytic differentiation of the human colon cancer cell line HT-29, which is differentiated in the absence (Glc-) but not in the presence of glucose (Glc+), have recently shown that the post-translational processing of sucrase-isomaltase and particularly its glycosylation vary as a function of cell differentiation (Trugnan G., Rousset, M., Chantret, I., Barbat, A., and Zweibaum, A. (1987) J. Cell Biol. 104, 1199-1205). Other studies indicate that in undifferentiated HT-29 Glc+ cells there is an accumulation of UDP-N-acetylhexosamine, which is involved in the glycosylation process (Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146). The purpose of the present work is to investigate whether an overall alteration of protein glycosylation is associated with the inability of HT-29 cells to differentiate. At least three alterations are detected: (i) after a 10-min pulse, the incorporation of D-[2-3H]mannose in undifferentiated cells is severely reduced, compared to differentiated cells. (ii) After a 24-h period of labeling with D-[2-3H]mannose, undifferentiated cells accumulate more than 60% of the radioactivity in the high mannose glycopeptides, whereas differentiated HT-29 Glc- cells accumulate only 38%. (iii) The analysis of the high mannose oligosaccharides transferred "en bloc" from the lipid precursor shows that Man9,8-GlcNAc2 species accumulate in undifferentiated cells, whereas no such accumulation can be detected in differentiated cells. This glycosylation pattern is consistent with an impairment of the trimming of high mannose into complex glycans. It is concluded that N-glycan processing is correlated with the state of enterocytic differentiation of HT-29 cells.

Control of the expression and activity of the Galpha-interacting protein (GAIP) in human intestinal cells.

The Galpha-interacting protein (GAIP) is known to interact with the Galphai3 protein. It has been suggested that, depending on its expression, GAIP can be a regulator of trimeric Gi protein signaling pathways. In the present study we show that the GAIP mRNA content declines during the enterocytic differentiation of two cell lines derived from human colon adenocarcinomas: HT-29 and Caco-2. In undifferentiated HT-29 cells, when the GDP/GTP cycle on the trimeric Gi3 protein is interrupted by either pertussis toxin treatment or by the transfection of a mutant of the Galphai3 protein with no GTPase activity (Q204L), we observed a decrease in the GAIP mRNA content. As these conditions are known to impair the Gi3-dependent lysosomal-autophagic pathway existing in undifferentiated HT-29 cells, we have investigated the role of GAIP in controling the lysosomal-autophagic pathway. Overexpression of GAIP stimulated protein degradation along the macroautophagic pathway. In contrast, overexpression of GAIP did not modify the low rate of macroautophagy in cells expressing the Q204L mutant of the Galphai3 protein. These results show that GAIP regulates a major catabolic pathway and that the expression of GAIP is dependent upon the activity of the Galphai3 protein and the state of enterocytic differentiation of cells.

Erk1/2-dependent phosphorylation of Galpha-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells.

Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells.

Autophagic degradation of N-linked glycoproteins is downregulated in differentiated human colon adenocarcinoma cells.

The aim of the present study was to elucidate the mechanism responsible for the high mannose glycoprotein instability in undifferentiated HT-29 cells (a human colon cancer cell line) reported previously. The results presented here are consistent with lysosomal degradation of these molecular species. In addition inhibitors of the autophagic-lysosomal degradative pathway (3-methyladenine, okadaic acid and asparagine) dramatically block the degradation of proteins and N-linked glycoproteins in undifferentiated HT-29 cells. The main conclusions of this work are: 1- the autophagic-lysosomal pathway is responsible for the high mannose glycoprotein degradation in undifferentiated HT-29 cells; 2- this degradative pathway exists in differentiated cells but is greatly reduced (3.5-4 fold); 3- the HT-29 cell line is a new model to investigate the molecular regulation of autophagy.

[Expression of O-glycans during enterocytic differentiation of HT-29 cells]. / Expression des O-glycannes au cours de la différenciation entérocytaire des cellules HT-29.

The expression of O- and N-glycan chains during the enterocytic differentiation of HT-29 cells was followed using L-[63H]-fucose and D-[6-3H]-glucosamine radiolabeling. Whatever the cell population, i.e., differentiated or undifferentiated, the incorporation of radiolabeled sugars into glycoproteins was similar. However, differences among these two cell populations were noted when the ratio [3H]-glucosamine/[3H]-galactosamine and the sensitivity of glycopeptides to mild alkaline treatment were followed. From these data, we could conclude that there is a shift in the high molecular weight glycopeptides during the differentiation of HT-29 cells meaning an increase of O-linked glycopeptides correlated with a decrease of N-linked forms.

Localization and processing of glycosylphosphatidylinositol anchored cathepsin D.

We have investigated the effect of a glycosylphosphatidylinositol anchor on the distribution of the soluble lysosomal enzyme cathepsin D. Only 10% of the chimeric protein (CD-GPI) could be detected on the plasma membrane after transfection in CHO cells. Similarly to endogenous cathepsin D, intracellular CD-GPI was detected in vesicular structures, suggesting that CD-GPI is targeted to lysosomes. CD-GPI is present as three forms with M(r) 55, 50 and 37 kD which could correspond to the precursor, intermediate and mature forms of cathepsin D, respectively. CD-GPI was shown to be GPI anchored by differential extractability with Triton X-114 before and after phosphatidylinositol phospholipase C hydrolysis. Intracellular CD-GPI is mainly substituted with oligosaccharides containing uncovered mannose 6-phosphate residues whereas these residues are covered in the cell surface precursor form of CD-GPI. Ammonium chloride treatment reduces the lysosomal delivery of CD-GPI and increases the cell surface expression of its precursor form.

Evidence for a dual control of macroautophagic sequestration and intracellular trafficking of N-linked glycoproteins by the trimeric G(i3) protein in HT-29 cells.

The trimeric G(i3) protein-dependent lysosomal-autophagic pathway is responsible for the degradation of a pool of N-linked glycoproteins in the human colon cancer HT-29 cell line. Here we have followed the fate of N-glycans using HT-29 cells either overexpressing the wild-type G alpha(i3) protein or transfected with different mutants of the G alpha(i3) protein. The stabilization of N-glycans was dependent upon the inhibition of autophagic sequestration by either 3-methyladenine (3-MA) or pertussis toxin (PTX). However, PTX allowed the processing of high-mannose glycans whereas 3-MA did not. The destabilization of the Golgi apparatus by brefeldin A, which interrupts the intracellular trafficking of N-linked glycoproteins along the secretory pathway, did not interfere with the macroautophagic pathway. These results suggest that the lysosomal-autophagic pathway is not dependent upon the integrity of the Golgi apparatus and points to differences between the molecular properties of two membrane flow processes (macroautophagy, exocytic pathway) controlled by the trimeric G(i3) protein.

Guanine nucleotide exchange on heterotrimeric Gi3 protein controls autophagic sequestration in HT-29 cells.

Recent results have shown that autophagic sequestration in the human colon cancer cell line HT-29 is controlled by the pertussis toxin-sensitive heterotrimeric Gi3 protein. Here we show that transfection of an antisense oligodeoxynucleotide to the alphai3-subunit markedly inhibits autophagic sequestration, whereas transfection of an antisense oligodeoxynucleotide to the alphai2-subunit does not change the rate of autophagy in HT-29 cells. Autophagic sequestration was arrested in cells transfected with a mutant of the alphai3-subunit (Q204L) that is restricted to the GTP-bound form. In Q204L-expressing cells, 3-methyladenine-sensitive degradation of long lived [14C]valine-labeled proteins was severely impaired and could not be stimulated by nutrient deprivation. Autophagy was also reduced when dissociation of the betagamma dimer from the GTP-bound alphai3-subunit was impaired in cells transfected with the G203A mutant. In contrast, a high rate of pertussis toxin-sensitive autophagy was observed in cells transfected with an alphai3-subunit mutant (S47N) which has an increased guanine nucleotide exchange rate and increased preference for GDP over GTP. Cells that express pertussis toxin-insensitive mutants of either wild-type alphai3-subunit (C351S) or S47N alphai3-subunit (S47N/C351S) exhibit a high rate of autophagy.

Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells.

3-Methyladenine which stops macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelárová, H., and Meijer, A. J. (1997) Eur. J. Biochem. 243, 240-246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3, 4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4, 5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced macroautophagy. In contrast, an increase in the class III PI3K product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates macroautophagy. Transfection of a specific class III PI3K antisense oligonucleotide greatly inhibited the rate of macroautophagy. In accordance with a role of class III PI3K, wortmannin (an inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC(50) 5-15 nM). Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the Galpha(i3) protein suggesting that both class III PI3K and trimeric G(i3) protein signaling are required in the control macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in macroautophagy are discussed in the context of membrane recycling.

Glucose persistence on high-mannose oligosaccharides selectively inhibits the macroautophagic sequestration of N-linked glycoproteins.

The macroautophagic-lysosomal pathway is a bulk degradative process for cytosolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell population is characterized by a high rate of autophagic degradation of N-linked glycoproteins substituted with ER-type glycans. In the present work we demonstrate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimycin] have a stabilizing effect on newly synthesized glucosylated N-linked glycoproteins and impaired their lysosomal delivery as shown by subcellular fractionation on Percoll gradients. The inhibition of macroautophagy was restricted to N-linked glycoproteins because macroautophagic parameters such as the rate of sequestration of cytosolic markers and the fractional volume occupied by autophagic vacuoles were not affected in CST-treated cells. The protection of glucosylated glycoproteins from autophagic sequestration was also observed in inhibitor-treated Chinese hamster ovary (CHO) cells and in Lec23 cells (a CHO mutant deficient in glucosidase I activity). The interaction of glucosylated glycoproteins with the ER chaperone binding protein (BiP) was prolonged in inhibitor-treated cells in comparison with untreated CHO cells. These results show that the removal of glucose from N-glycans of glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glucosylated N-linked glycoproteins from being sequestered by the autophagic pathway.

Subcellular localization of the Galphai3 protein and G alpha interacting protein, two proteins involved in the control of macroautophagy in human colon cancer HT-29 cells.

Autophagic sequestration is controlled by the Galphai3 protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the Galphai3 protein is preferentially associated with Golgi membranes but co-localization was also observed with the endoplasmic reticulum (ER) membrane. The Galphai2 protein, which is not involved in the control of autophagic sequestration, is associated with the plasma membrane. Transfection of chimaeric Galphai proteins (Galphai3/2, Galphai2/3) containing the N- and C-terminal parts of the relevant Galphai demonstrated that the C-terminal part of the Galphai3 protein, by governing its membrane localization [de Almeida, Holtzman, Peters, Ercolani, Ausiello and Stow (1994) J. Cell Sci. 107, 507-515], is important in the control of macroautophagic sequestration. G alpha interacting protein (GAIP),which stimulates the GTPase activity of the Galphai3 protein and favours macroautophagic sequestration in HT-29 cells,was shown, by immunofluorescence studies using confocal microscopy, to be confined to the cytoplasm. The cytoplasmic distribution of GAIP only partially overlaps with that of the Galphai3 protein. However, the presence of the two proteins on Golgi and ER membranes was confirmed by subcellular fractionation. These results point to the importance of the cytoplasmic localization of the Galphai3 protein and GAIP in controlling autophagic sequestration in HT-29 cells.

WGA binding to the surface of two autologous human melanoma cell lines: different expression of sialyl and N-acetylglucosaminyl residues.

Two autologous human melanoma cell lines were studied to determine their capacities to bind wheat germ agglutinin (WGA). Both cell lines were derived from the same patient, the first, IGR 39, originated from the primary tumor, the second, IGR 37, was established from a metastatic lymph node. WGA binding sites on the surface of these cell lines were compared before and after sialidase and/or tunicamycin treatments. IGR 39 cells exhibited two classes of WGA binding sites with high and low affinities, whereas IGR 37 cells had only one class of high affinity binding sites. After tunicamycin treatment, the capacity of IGR 39 cells to bind WGA was markedly altered, since only one class of WGA binding sites with high affinity was observed under these conditions, whereas tunicamycin did not induce significant changes in the lectin binding of IGR 37 cells. The low affinity WGA binding sites, which were only found on IGR 39 cells, corresponded to sialyl residues present in N-linked glycoproteins. The high affinity binding sites present on both cell lines probably involved sialyl and N-acetyl-glucosaminyl residues associated with O-linked glycoproteins and/or glycolipids. No direct correlation could be drawn between the number of WGA binding sites and the overall sialic acid levels exposed to sialidase treatment. The 3-fold increase in the amount of cell surface glycopeptides obtained after pronase digestion and specifically binding to WGA-Sepharose was in good agreement with the overall higher number of WGA binding sites on IGR 39 compared to IGR 37 cells. Thus, subtle carbohydrate changes of cell surface glycoconjugates might account for the differences between the biological properties of human melanoma cell lines of low and high tumorigenicity.