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PubMed2002

Oxidation of the glutathione/glutathione disulfide redox state is induced by cysteine deficiency in human colon carcinoma HT29 cells.

Glutathione (GSH) has a central role in the maintenance of the thiol-disulfide redox state in mammalian cells. GSH synthesis can be physiologically limited by the availability of cysteine (Cys), and Cys and its precursors are variable in the human diet. The purpose of this study was to determine the effect of severe Cys deficiency and readdition of Cys on the redox state of the GSH/glutathione disulfide (GSSG) pool in human colon carcinoma HT29 cells. Cells were cultured in Cys- (and cystine-)limiting medium for 48 h followed by culture in medium containing either Cys or cystine for 24 h. GSH and GSSG were measured by HPLC. Cys limitation decreased cellular GSH and GSSG concentrations with an associated >80 mV oxidation of the GSH/GSSG redox state. Upon addition of either Cys or its disulfide cystine (CySS), redox of GSH/GSSG recovered in 4 h, whereas GSH concentration continued to increase over 12 h. Maximal GSH concentrations attained were 200% of control cell values. These results show that severe Cys deficiency can have marked effects on cellular redox state but that redox recovers rapidly upon resupply. The magnitude of oxidation during Cys limitation in this cell model is sufficient to result in a >100-fold change in the reduced/oxidized ratio of redox-sensitive dithiol/disulfide motifs in proteins. If redox changes occur in vivo in association with variations in dietary Cys and its precursors, these changes could have important physiologic effects through altered redox signaling and control of cell proliferation and apoptosis.

Colonic Neoplasms↗
PubMed1993

Agonist-induced intracellular Ca2+ transients in HT29 cells.

In the present study we have investigated the mechanism of intracellular Ca2+ activity ([Ca2+]i) changes in HT29 cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1-40Hz). ATP and CCH induced not only a dose-dependent [Ca2+]i peak response, but also changes of the plateau phase. The [Ca2+]i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca2+]i plateau increased at higher CCH concentrations. NT showed (from 10(-10) to 10(-7) mol/l) in most cases only a [Ca2+]i spike lasting 2-3 min. The [Ca2+]i plateau induced by ATP (10(-6) mol/l) and CCH (10(-5) mol/l) was abolished by reducing the Ca2+ activity in the bath from 10(-3) to 10(-4) mol/l (n = 7). In Ca(2+)-free bathing solution the [Ca2+]i peak value for all three agonists was not altered. Using fura-2 quenching by Mn2+ as an indicator of Ca2+ influx the [Ca2+]i peak was always reached before Mn2+ influx started. Every agonist showed this delayed stimulation of the Ca2+ influx with a lag time of 23 +/- 1.5 s (n = 15) indicating a similar mechanism in each case. Verapamil (10(-6)-10(-4) mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca2+]i increase. Short pre-incubation with verapamil augmented the effect on the [Ca2+]i peak, whereas no further influence on the plateau was observed. Ni2+ (10(-3) mol/l) reduced the plateau value by 70%.

Adenosine Triphosphate↗
PubMed1989

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells.

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.

Binding, Competitive↗
PubMed2005

Inhibitory effect on TNF-alpha-induced IL-8 production in the HT29 cell of constituents from the leaf and stem of Weigela subsessilis.

Twelve compounds were isolated from the MeOH extract of the leaf and stem of the Korean endemic plant Weigela subsessilis L. H. Bailey. Their chemical structures were elucidated on the basis of physicochemical and spectroscopic data and by comparison with those of published literatures. These compounds were identified as three sterols, beta-sitosterol acetate (2), betasitosterol (3), daucosterol (11), eight triterpenoids, squalene (1), ursolic acid (4), ilekudinol A (5), corosolic acid (6), ilekudinol B (7), esculentic acid (8), pomolic acid (9), asiatic acid (10), and one iridoid glycoside, alboside I (12). This is the first report pertaining to the isolation of these compounds from Weigela subsessilis L. H. Bailey. In addition, three compounds 7, 9, and 12 were found to display a strong inhibitory effect on the production of IL-8 in the HT29 cells stimulated by TNF-alpha.

Caprifoliaceae↗
PubMed1988

Characterization and possible mechanisms of alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production in HT29 cells.

Preincubation with an alpha 2-adrenergic agonist sensitized subsequent forskolin- and vasoactive intestinal peptide-stimulated cyclic AMP production in HT29 cells, a human colonic adenocarcinoma cell line. Preincubation with somatostatin, another agonist negatively coupled to adenylate cyclase, sensitized forskolin-stimulated cyclic AMP production to a lesser extent. alpha 2-Adrenergic agonist preincubation also resulted in desensitization as indicated by a shift to the right in the dose-response curve of a subsequent challenge by an alpha 2-adrenergic agonist. In an effort to elucidate the mechanism for sensitization, we examined protein kinase C and the Na+/H+ antiporter. Whereas these components had marked effects on forskolin stimulation, there was no effect on sensitization. Changes in the concentration of extra-cellular Ca2+ or Mg2+ had no effect on either forskolin stimulation or sensitization. Pertussis toxin pretreatment caused a time-dependent decrease in sensitization, an attenuation of inhibition of cyclic AMP production, and a decrease in subsequent [32P]ADP-ribosylation by pertussis toxin. The time course for these three events was similar, implicating the inhibitory guanine nucleotide regulatory protein in the mechanism for alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production. In addition, pertussis toxin dramatically decreased forskolin-stimulated cyclic AMP production, although with a different time course. These results suggest that the mechanism of sensitization is via an as yet undefined sequence of biochemical events that includes the inhibitory guanine nucleotide regulatory protein, but does not include inhibition of adenylate cyclase nor activation of the Na+/H+ antiporter.

Adenocarcinoma↗
PubMed2004

Expression of the hereditary hemochromatosis protein HFE increases ferritin levels by inhibiting iron export in HT29 cells.

Iron is essential for life in almost all organisms and, in mammals, is absorbed through the villus cells of the duodenum. Using a human colonic carcinoma cell line that has many duodenal characteristics, HT29, we show that genes involved in intestinal iron transport are endogenously expressed. When stably transfected to express the hereditary hemochromatosis protein HFE these cells have increased ferritin levels. We demonstrate that this is not due to an effect on the transferrin (TF)-mediated iron uptake pathway but rather due to inhibition of iron efflux from the cell. The effect of HFE was independent of its interaction with TF receptor 1 as indicated by similar results using both the wild type HFE and the W81A mutant that binds TF receptor 1 with greatly reduced affinity. HFE expression did not affect the mRNA levels of most of the genes involved in iron absorption that were tested; however, it did correspond to a decrease in hephaestin message levels. These results point to a role for HFE in inhibition of iron efflux in HT29 cells. This is a distinct role from that in HeLa and human embryonic kidney 293 cells where HFE has been shown to inhibit TF-mediated iron uptake resulting in decreased ferritin levels. Such a distinction suggests a multifunctional role for HFE that is dependent upon expression levels of proteins involved in iron transport.

Antigens, CD↗
PubMed2000

Synergistic antitumoral activity of combined UFT, folinic acid and oxaliplatin against human colorectal HT29 cell xenografts in athymic nude mice.

This study was designed to assess the inhibition of tumor growth by oxaliplatin combined with UFT and folinic acid (FA). Growth inhibition was studied in nude mice transplanted with human colorectal HT29 tumor cell xenografts and treated for 28 days with oral UFT (20 mg/kg/day) and FA (4 mg/kg/day), i.p. oxaliplatin (10 mg/kg on day 1) or a combination of oxaliplatin, UFT and FA, or else not treated (controls). Tumor surface area and weight were recorded twice a week, and mice were sacrificed at day 28. Two separate experiments were performed for each group of 25 mice. At day 28, mean tumor weights (g) were 2.89+/-0.22 (controls), 2.03+/-0.14 (oxaliplatin), 2.02+/-0.21 (UFT/FA) and 1.23+/-0.17 (oxaliplatin+UFT/FA). For the three treatment groups, tumor weight decreases were 30.1% (p<0.05), 29.9% (p<0.05) and 57.5% (p<0.001), respectively. Combined treatment (UFT/FA+oxaliplatin) reduced tumor weight by 39% compared to oxaliplatin alone (p<0.05) or UFT/FA (p<0.05). These results demonstrate the synergistic effect of the combination of oxaliplatin, UFT and FA in this HT29 cell xenograft model, and warrant further investigations in patients with metastatic colorectal cancer.

Animals↗
PubMed1990

Effects of alpha 2-adrenergic agonist preincubation on subsequent forskolin-stimulated adenylate cyclase activity and [3H]forskolin binding in membranes from HT29 cells.

alpha 2-Adrenergic agonist preincubation resulted in a leftward shift in the subsequent concentration-response curve to forskolin-stimulated adenylate cyclase activity in membranes from HT29 cells, a human colon adenocarcinoma cell line. This effect was much less pronounced than the effect seen in the intact cell cyclic AMP production assays. Removal of GTP from the assay caused a further slight leftward shift in the concentration-response curve. In [3H]forskolin binding experiments, alpha 2-adrenergic agonist preincubation caused a doubling of the maximal number of binding sites (80 vs 31 fmol/mg protein) compared to control. The addition of MgCl2 and NaF to the assay buffer increased control binding 5-fold. With agonist preincubation, there was a further increase in binding in the presence of MgCl2 and NaF which was not significantly different from the appropriate control. Pertussis toxin pretreatment blocked both the leftward shift in the forskolin concentration-response curve and the increase in maximal number of binding sites, indicating that a pertussis toxin sensitive protein is involved in these changes. Activation of cyclic AMP production in the intact cell by cholera toxin followed by norepinephrine preincubation and then stimulation by forskolin resulted in a degree of sensitization similar to that seen in the membrane adenylate cyclase and binding assays. Pertussis toxin also blocked this sensitization. It appears that if the cyclase system is highly activated, then the degree of sensitization is similar in the membrane and intact cell assay.

Adenylate Cyclase Toxin↗
PubMed1993

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling.

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca2+ activity ([Ca2+]i) in HT29 cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca2+]i to a stable plateau of 10-20 nmol/l above resting [Ca2+]i was observed. Lower osmolalities resulted in a triphasic increase of [Ca2+]i to a peak value. In a first phase after the volume change, lasting 20-40 s, [Ca2+]i increased slowly by about 30 nmol/l. Thereafter [Ca2+]i increased more rapidly within 20-30 s to a peak value. This peak was 189 +/- 45 nmol/l (190 mosmol/l, n = 9) and 243 +/- 41 nmol/l (160 mosmol/l, n = 20) above resting [Ca2+]i. The peak was then followed by a decline of [Ca2+]i over the next 2-3 min to a stable plateau value of 28 +/- 6 (n = 6) and 32 +/- 11 nmol/l (n = 11) above resting [Ca2+]i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca(2+)-free bath conditions the peak value for the cell-swelling-induced [Ca2+]i transient was reached significantly later (60-100 s, compared to 40-60 s under control conditions). The peak values under Ca(2+)-free conditions were not significantly lower. This indicates that the [Ca2+]i peak was mostly of intracellular origin. No [Ca2+]i plateau phase was observed under Ca(2+)-free bath conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1988

Blocking kinetics of Cl- channels in colonic carcinoma cells (HT29) as revealed by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB).

The blocking effect of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was investigated on single Cl- channels of the cultured human colon carcinoma cells, HT29. In the absence of NPPB, the open-time histogram yielded two time constants, with 0.9 ms and 33 ms, whereas the closed-time distribution could be fitted by a single exponential with a time constant of 0.7 ms. Addition of NPPB in the range 1-50 microM induced brief closing events of the single-channel current. This resulted in a decrease of the long open-time constant to 2.1 ms and in an increase of the closed-time constant to 1.8 ms at 50 microM NPPB concentration. The short open-time constant did not change at low blocker concentration (1 microM), but could no longer be resolved at higher concentrations. The open-state probability decreased from 0.9 (control conditions) to 0.5 at 50 microM NPPB. The Hill plot yielded a Hill coefficient of about 0.7, compatible with one NPPB molecule inhibiting one channel molecule. The kinetics of channel gating are described by a sequential model with one closed and two open states. Since in the presence of NPPB no additional time constant appeared in the time histograms, we assumed the same kinetic scheme as under control conditions, and hypothesize that NPPB has an influence on rate constants.

Cell Line↗
PubMed2005

Effects of Brussels sprout juice on the cell cycle and adhesion of human colorectal carcinoma cells (HT29) in vitro.

Consumption of Brassica vegetables is associated with a reduced risk of cancer of the alimentary tract in animal models and human populations. We used raw juice extracted from Brussels sprouts rich in the glucosinolate sinigrin to explore the effect of naturally occurring glucosinolate breakdown products on cell cycle progression and apoptosis in human colorectal carcinoma cells (HT29). Juice was prepared from sprout tissue immediately before use, and the glucosinolate breakdown products were determined by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The cell cycle was analyzed by flow cytometry on detached and adherent cells, and apoptosis was measured in the detached population by annexin V staining. Twenty-four hours after challenge with juice (10 microL/mL), 7-13% of adherent cells had detached from the substratum but the majority (82%) of these cells had not entered apoptosis, whereas only 33% of detached control cells were not apoptotic (p < 0.05). The main glucosinolate breakdown products were as follows: the sinigrin breakdown product, 1-cyano-2,3-epithiopropane (ca. 38 mM); the gluconapin hydrolysis product, 3-butenyl isothiocyanate (ca. 2.2.mM); the glucobrassicin metabolite, ascorbigen (ca. 8 mM); and low concentrations of other indole glucosinolate-derived hydrolysis products such as neoascorbigen and 3,3'-diindolylmethane. A variety of biologically active glucosinolate breakdown products are released by mechanical disruption of raw Brussels sprout tissue, but contrary to previous assumptions, allyl isothiocyanate is not the main compound responsible for the inhibition of cell proliferation.

Apoptosis↗
PubMed1987

Characteristics of apical chloride channels in human colon cells (HT29).

Recent studies have demonstrated that active chloride secretion in mammalian colon and other epithelia, is dependent on the induction of an increase of apical chloride conductance. Since the physical characteristics of apical chloride channels in man have not been elucidated, patch clamp analysis of human colon cells (HT29), in culture, was performed, after stimulation with db-cAMP 10(-4) mol/l. In excised inside out patches of apical membranes two types of channels were found. The smaller and less frequent channel had a mean conductance of 15 +/- 1 pS (n = 9). This type of channel showed identical I/V curves in NaCl and KCl solutions. It was inhibited by a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The more frequently observed larger conductance channel was selective for anions and was impermeable to Na+ and K+. Regarding anion selectivity, the channel was similarly permeable to Cl-, Br-, I-, and NO3-, but was impermeable to gluconate. The channel was completely inhibited by the potent Cl- channel blocker NPPB (10(-6) mol/l). This channel exhibited rectification: The conductance was 50 +/- 4 pS at positive clamp potentials (sign referred to bath with respect to pipette interior) and 32 +/- 3 (n = 33) pS at negative voltages. Moreover, the open state probability was doubled when the clamp potential was increased from -20 to +20 mV. These results demonstrate the existence of chloride channels in the apical membrane of db-cAMP treated colonic carcinoma cells.

Chlorides↗
PubMed1993

Differential metabolism and trafficking of sphingolipids in differentiated versus undifferentiated HT29 cells.

Trafficking and metabolism of sphingolipids were examined in undifferentiated (G+) and differentiated (G+ reversed) HT29 human colon adenocarcinoma cell lines. Metabolic experiments employing a fluorescently labeled sphingolipid precursor, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylceramide++ + (C6-NBD-ceramide) revealed that both qualitative and quantitative differences exist in sphingolipid synthesis between the 2 cell lines. One of the C6-NBD-sphingolipids synthesized in G+ cells is not found in the G+ reversed cells. Furthermore, the ratio of the 2 main products, C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, differs: in G+ cells glucosylceramide is by far the main product, whereas G+ reversed cells synthesize C6-NBD-sphingomyelin in slight excess. Once established, these ratios of sphingolipids are quickly restored metabolically when distortion of the ratio is caused by experimental manipulation. This indicates that they represent a true metabolic equilibrium situation of the 2 sphingolipids in these cells, while the distinct ratios are mainly determined by the NBD-lipid pool in the plasma membrane. Preferential synthesis and transfer of glucosylceramide from its site of synthesis to the cell surface do not occur when the plasma membrane pool of glucosylceramide is selectively removed. This suggests that instantaneous replenishment via specific signalling is probably not involved as a mechanism in re-establishing perturbed lipid pools. In conjunction with observations on distinct lipid trafficking pathways of glucosylceramide in G+ and G+ reversed cells, the present metabolic studies emphasize a relation between the expression of this glycolipid and the state of differentiation of HT29 cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

Apoptosis in human adenocarcinoma HT29 cells induced by exposure to hypoxia.

BACKGROUND: Recent evidence indicates that programmed cell death or apoptosis may be an important mechanism for the lethality of cancer chemotherapy drugs in tumor cells. Apoptosis may be induced in tumor cells by a number of physical stresses, including radiation, UV light, heat shock, and cold. In preliminary studies, we have found that exposure of cells to hypoxia rapidly induces the expression of several immediate early genes, including c-jun, jun-D, and c-fos, and of a bifunctional redox protein/endonuclease, Ref-1. PURPOSE: Our purpose was to determine if hypoxia-induced overexpression of the endonuclease was associated with altered DNA integrity and manifestations of apoptosis. METHODS: We examined cultured cells for the induction of apoptosis by electrophoresis and immunofluorescence techniques and related these findings to the induction of gene expression by hypoxia. RESULTS: We found that an 8-hour exposure of human adenocarcinoma HT29 cells to hypoxia (which results in only 14% loss of viability) induced internucleosomal DNA fragmentation beginning after 8 hours of hypoxia and before reoxygenation. Immunofluorescence of hypoxia-treated cells showed that apoptotic cells were detectable initially after 4 hours of hypoxia and reached a peak (31.3% of cells) at 12 hours after reoxygenation and that by 36 hours after reoxygenation all apoptotic cells had been eliminated from the population. Hypoxia-induced apoptosis was associated with a marked induction of c-myc messenger RNA (mRNA). We have previously shown that hypoxia is associated with ref-1 mRNA induction. While expression of c-myc declined with a time course similar to that of the disappearance of apoptotic cells, that of ref-1 remained elevated. Coincident with the decline of c-myc, we observed a late increase in the expression of bcl-2 beginning 24 hours after reoxygenation. CONCLUSIONS AND IMPLICATIONS: These results suggest a possible relationship between Ref-1 induction and the occurrence of apoptosis following a hypoxic exposure.

Adenocarcinoma↗
PubMed2001

Induction of apoptosis in human colon carcinoma cells HT29 by sublethal cryo-injury: mediation by cytochrome c release.

Cryosurgery is an emerging treatment for human solid tumors, notably colorectal liver metastasis. Cryosurgical procedures generate a thermal gradient of from at least -50 degrees C at the center of the tumor being treated to about 0 degrees C at the periphery. Cell death occurs by necrosis in the center, while the peripheral zone of frozen tumor harbors a mix of viable and dead tissue. In order to understand the mechanisms of cell death and survival in this peripheral area at risk for tumor recurrence, we have established an in vitro freezing system that mimics in vivo conditions of sublethal injury. HT29 colon cancer cells were subjected to freezing temperatures from -6 degrees C to -36 degrees C, thawed at room temperature for 30 min and rewarmed at 37 degrees C for a period of time. Post-freeze-thaw, cryolytic cells were evaluated by trypan blue exclusive assay. We also identified apoptotic cells after rewarming by cell shrinkage, nucleic condensation, TUNEL assay, DNA fragmentation and PARP degradation. The intensity of cryolysis and apoptosis was increased by lowering the freezing temperature. At -36 degrees C, all cells were dead immediately after freeze-thaw. A kinetic analysis of cryo-induced apoptosis showed that the commitment to enter apoptosis occurred right after the freeze-thaw period and lasted less than 8 hr after rewarming. We further demonstrated that freezing triggers one of the caspase cascade involved in apoptosis: release of cytochrome c from mitochondria to cytosol, followed by activation of caspase-9 and degradation of PARP. These results indicate the death of cancer cells under cryo-treatment at sublethal freezing temperature can be attributed 2 different modes, cryolysis as well as apoptosis. HT29 cells carrying p53 mutant have very quick response for induction of apoptosis by cryo-treatment and contain an intact pathway of caspase cascade. Further studies will address if mechanisms in cells with wild-type p53 will differ.

Apoptosis↗
PubMed1996

Dependence of fluorodeoxyuridine-induced cytotoxicity and megabase DNA fragment formation on S phase progression in HT29 cells.

The relationship between cell cycle progression and induction of DNA double-strand breaks and cytotoxicity by exposure to fluorodeoxyuridine (FdUrd) was studied in HT29 human colon cancer cells. Fractionation of drug-treated populations by centrifugal elutriation yielded subpopulations having widely divergent abilities to progress through S phase in the presence of the drug. One of these subpopulations, which appeared to undergo coordinated growth arrest, was resistant to FdUrd cytotoxicity and DNA damage. In contrast, the subpopulation which was able to progress furthest through S phase in the presence of FdUrd underwent unbalanced growth arrest (i.e., increase in size and mass out of proportion to DNA synthesis), and displayed both DNA double-strand break formation (assayed by pulsed field gel electrophoresis) and loss of clonogenicity. When cells were elutriated prior to drug treatment, producing fractions enriched in cells at various cell cycle stages, no significant differences in sensitivity to FdUrd-induced cytotoxicity were detected among elutriation fractions. These findings support the model that, in HT29 cells, progression into and through S phase during drug treatment is an important determinant of FdUrd-induced DNA damage and cytotoxicity, but that the cell cycle position at the start of drug exposure is not a critical factor for these effects.

Antimetabolites, Antineoplastic↗
PubMed1994

Transport of biosynthetic sphingolipids from Golgi to plasma membrane in HT29 cells: involvement of different carrier vesicle populations.

Intracellular transport of the sphingolipids glucosylceramide (GlcCer) and sphingomyelin (SM), was examined in HT29 human colon adenocarcinoma cells. After synthesis from a fluorescent precursor, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylceramide++ + (C6-NBD-Cer), transfer of SM from the Golgi complex to the plasma membrane can occur independently of that of GlcCer, as revealed by temperature-dependent experiments. Thus, at 20 degrees C, SM trafficking to the cell surface is essentially unaffected, whereas GlcCer transport to the plasma membrane is inhibited by approximately 75%, when compared to the transfer of both lipids at 37 degrees C. The mechanism by which SM and GlcCer are transported to the cell surface involves at least in part a vesicular mechanism. Transport vesicles, containing both lipids at their luminal surface, as revealed by the inaccessibility of the NBD fluorescence to the quencher sodium dithionite, have been isolated from cells, permeabilized by filter stripping. As evidenced by electron microscopic and biochemical criteria, no vesicles or lipids were released when cell permeabilization had been carried out with streptolysin. Density gradient analysis indicates the potential existence of several vesicle populations, distinctly enriched in either lipid, involved in transport of sphingolipids to the plasma membrane in HT29 cells.

Adenocarcinoma↗