The posttranslational processing of sucrase-isomaltase in HT-29 cells is a function of their state of enterocytic differentiation

The biosynthesis of sucrase-isomaltase was compared in enterocyte-like differentiated (i.e., grown in the absence of glucose) and undifferentiated (i.e., grown in the presence of glucose) HT-29 cells. Unlike differentiated cells, in which the enzyme is easily detectable and active, undifferentiated cells display almost no enzyme activity and the protein cannot be detected by means of cell surface immunofluorescence or immunodetection in membrane-enriched fractions or cell homogenates. Pulse experiments with L-[35S]-methionine show that the enzyme is, however, synthesized in these undifferentiated cells. As compared with the corresponding molecular forms in differentiated cells, the high-mannose form of the enzyme in undifferentiated cells is similarly synthesized and has the same apparent Mr. However, its complex form is less labeled and has a lower apparent Mr. Pulse-chase experiments with L-[35S]methionine show that, although the enzyme is synthesized to the same extent in both situations, the high-mannose and complex forms are rapidly degraded in undifferentiated cells, with an apparent half-life of 6 h, in contrast to differentiated cells in which the enzyme is stable for at least 48 h. A comparison of the processing of the enzyme in both situations shows that the conversion of the high-mannose to the complex form is markedly decreased in undifferentiated cells. These results indicate that the absence of sucrase-isomaltase expression in undifferentiated cells is not the consequence of an absence of biosynthesis but rather the result of both an impaired glycosylation and a rapid degradation of the enzyme.     

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.     

Swainsonine is a useful tool to monitor the intracellular traffic of N-linked glycoproteins as a function of the state of enterocytic differentiation of HT-29 cells.

After treatment with swainsonine, an inhibitor of both lysosomal alpha-mannosidase and Golgi alpha-mannosidase-II activities, analysis of [3H]mannose-labeled glycans showed that HT-29 cells, derived from a human colonic adenocarcinoma, displayed distinct patterns of N-glycan expression, depending upon their state of enterocytic differentiation. In differentiated HT-29 cells hybrid-type chains were detected, whereas undifferentiated HT-29 cells accumulated high-mannose-type oligosaccharide, despite our demonstration of Golgi alpha-mannosidase-II activity in both cell populations. Pulse/chase experiments carried out in the presence of swainsonine revealed that the persistence of high-mannose-type chains in undifferentiated HT-29 cells was the result of the stabilization of glycoproteins substituted with these glycans. These data suggest that in undifferentiated HT-29 cells, glycoproteins with high-mannose-type oligosaccharides are delivered to a degradative compartment containing swainsonine-sensitive alpha-mannosidase(s), whereas in differentiated HT-29 cells glycoproteins enter a compartment in which alpha-mannosidase II (Golgi apparatus) is present. Thus, this apparent dual effect of swainsonine on N-glycan trimming may reflect differences in the intracellular traffic of glycoproteins as a function of the state of enterocytic differentiation of HT-29 cells.     

N-glycosylation modification of proteins is an early marker of the enterocytic differentiation process of HT-29 cells

The human colon cancer cell line HT-29 remains totally undifferentiated when glucose is present in the culture medium (HT-29 Glc+), while the same cells may undergo typical enterocytic differentiation after reaching confluence when grown in glucose-deprived medium (HT-29 Glc-). Recently, we demonstrated a deficiency in the overall N-glycan processing in confluent undifferentiated cells, whereas differentiated cells follow a classical pattern of N-glycosylation. The main changes in N-glycosylation observed in confluent undifferentiated cells may be summarised as follows: 1) the conversion of high mannose into complex glycopeptides is greatly decreased; 2) this decreased conversion could be a consequence of an accumulation of Man9-8-GlcNAc2-Asn high mannose species. Whether these changes in N-glycan processing appear progressively during cell culture or are already present from the beginning of the culture was investigated in this study by comparing the actual status of N-glycan processing in exponentially growing HT-29 Glc- and HT-29 Glc+ cells. Under these conditions, HT-29 Glc- cells do not exhibit any characteristics of differentiation. The conversion of high mannose into complex glycoproteins is severely reduced in HT-29 Glc+ cells, regardless of the growth phase studied. In contrast, HT-29 Glc- cells display a normal pattern of N-glycan processing in both growth phases. We therefore conclude that N-glycan processing may be used as an early biochemical marker of the enterocytic differentiation process of HT-29 cells.     

Energy state in HT-29 cells is linked to differentiation

Summary The relationship between the energy source used by HT-29 cells and their state of differentiation was determined. Short chain fatty acids and acetoacetate were applied to the cells for 9 d, after which the medium was replaced with conventional culture medium for a further 9 d so that the permanence of the changes could be assessed (18 d). Glucose utilization and lactic acid, acetoacetate, and β-hydroxybutyrate production by the cells were determined. Differentiation was assessed by the presence of the enzymes sucrase-isomaltase and carbonic anhydrase 1, as well as morphological changes of the cells. By tracing carbon from acetate, propionate, and butyrate through the cells, it was found that the carbon from the short-chain fatty acids was fluxed into acetoacetate. Significant amounts of acetoacetate were released by the propionate-treated culture after 9 d and the acetate-, propionate-, valerate-, and caproate-treated cultures after 18 d. A significant positive correlation was found between acetoacetate synthesis and differentiation. Acetoacetate applied to HT-29 cells also induced their differentiation. The acetate-, butyrate-, valerate-, isovalerate-, and caproate-treated cells underwent terminal differentiation, while the propionate- and isocaproate-treated cultures underwent programming events. We, therefore, conclude that HT-29 cells utilize short chain fatty acids in preference to glucose, metabolize these to ketones, thereby raising the energy state and effecting the observed morphological and functional changes in the cells.     

Activity and properties of alpha-L-fucosidase are dependent on the state of enterocytic differentiation of HT-29 colon cancer cells

Previously we have demonstrated an impairment in the activity of alpha-L-fucosidase in colon tumours. In order to establish an in vitro model to study this enzyme in colon cancer, we have determined the activity and properties of the enzyme during the differentiation of HT-29 colon cancer cells. Cultures were committed to differentiate into enterocyte-like cells by placing them in a culture medium without glucose for 18-21 days. The state of differentiation was evaluated by assaying the activity of enterocytic marker enzymes, and the acquisition of enterocyte morphology was assessed by electron microscopy. The alpha-L-fucosidase activity was determined using a fluorometric method. Intracellular levels of alpha-L-fucosidase activity are lower in non-differentiated cells (3.0 +/- 1.01 U/mg) than in differentiated ones (9.2 +/- 4.09 U/mg) (P < 0.001). This variation is not due to a greater secretion of the enzyme to the culture medium, and properties such as pH optimum or the affinity towards substrate are not dependent on differentiation. The enzyme however, is more stable at acidic pH and at high temperatures, and V(max) is higher in differentiated cells. Moreover, in undifferentiated cells the enzyme is mainly in a monomeric form whereas multimeric forms of the enzyme appear only upon differentiation. Most of these changes are very similar to those previously observed between normal colon tissue and colon tumours. Thus, we suggest that differentiation of HT-29 colon cancer cells could be used as a model to study the alterations of the enzyme alpha-L-fucosidase during the progression of the tumoural process.     

Docosahexaenoic acid is a potent inducer of apoptosis in HT-29 colon cancer cells

Some studies have shown that dietary intake of polyunsaturated fatty acids of the n-3 series may have inhibitory effect on the growth of tumor cells both in vivo and in vitro. However, the cellular and molecular mechanisms by which n-3 fatty acids reduce the growth of tumor cells remain poorly understood. In the present studies, we compared the potency of a variety of n-3 and n-6 fatty acids in modulating the apoptotic cell death in HT-29 colon cancer cells. Of all fatty acids examined, we found that docosahexaenoic acid (22:6n-3; DHA) is a potent inducer of apoptosis in a time- and dose-dependent manner. Indomethacin, a cyclooxygenase inhibitor, is ineffective in blocking the apoptosis induced by DHA, suggesting that DHA-induced apoptosis in HT-29 cells is not mediated through the cyclooxygenase pathway. In contrast, the DHA-induced apoptosis is partially reversed by a synthetic antioxidant, butylated hydroxytoluene, indicating that lipid peroxidation may be involved in apoptotic signaling pathway induced by DHA. DHA treatment decreased bcl-2 levels in association with apoptosis, whereas bax levels remained unchanged. These results suggest that decreased expression of bcl-2 by DHA might increase the sensitivity of cells to lipid peroxidation and to programmed cell death.     

Cytokine-induced apoptosis in epithelial HT-29 cells is independent of nitric oxide formation. Evidence for an interleukin-13-driven phosphatidylinositol 3-kinase-dependent survival mechanism.

A combination of the pro-inflammatory cytokines interleukin (IL)-1alpha, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997) J. Biol. Chem. 272, 12626-12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-gamma/TNF-alpha, IFN-gamma/CD95, IL-1alpha/IFN-gamma, and IL-1alpha/IFN-gamma/TNF-alpha to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1alpha/IFN-gamma/TNF-alpha- and IFN-gamma/TNF-alpha- as well as IFN-gamma/CD95-induced (but not IL-1alpha/IFN-gamma-induced) cell death. In addition, IFN-gamma/TNF-alpha and IL-1alpha/IFN-gamma/TNF-alpha stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.     

STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells.

Embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). LIF acts through a receptor complex composed of a low affinity LIF receptor (LIFRbeta) and gp130. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 revealed that the tyrosine residue of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we constructed a fusion protein composed of the entire coding region of STAT3 and the ligand binding domain of the estrogen receptor. This construction (STAT3ER) induced expression of junB (one of the targets of STAT3) in ES cells in the presence of the synthetic ligand 4-hydroxytamoxifen (4HT), thereby indicating that STAT3ER is a conditionally active form. ES cells transfected with STAT3ER cultured in the presence of 4HT maintained an undifferentiated state. Taken together, these results strongly suggest that STAT3 activation is required and sufficient to maintain the undifferentiated state of ES cells.     

Development of vasoactive intestinal peptide-responsive adenylate cyclase during enterocytic differentiation of Caco-2 cells in culture. Evidence for an increased receptor level

The purpose of this work was to study vasoactive intestinal peptide (VIP) receptors and the adenylate cyclase response to VIP upon enterocytic differentiation of the human colon adenocarcinoma cell line Caco-2 in culture. The VIP-stimulated enzyme activity is very low, e.g. 20% above basal activity in undifferentiated cells (day 5) and is enhanced markedly at confluency reaching a maximum, e.g. 270%, above basal activity in fully differentiated cells (day 30). VIP potency is also slightly enhanced, the EC50 of VIP ranging from 0.31 nM at day 5 to 0.07 nM at day 30. Modifications of the adenylate cyclase system are not responsible for the development of VIP response. Indeed, forskolin-stimulated adenylate cyclase activity is unchanged during differentiation supporting no alteration of the enzyme catalytic subunit. The same holds true for NaF and guanosine 5'-(beta, gamma-imido)trisphosphate, indicating a constant activity of the guanine nucleotide regulatory unit which mediates hormonal stimulation of adenylate cyclase (Ns). This is further supported by the similar extent of cholera toxin-catalyzed [32P]ADP-ribosylation of the Ns protein that is observed during differentiation. In sharp contrast, a dramatic increase of VIP receptor concentration is observed ranging from 32 fmol/mg of protein at day 5 to 414 fmol/mg of protein at day 30. This is confirmed by affinity cross-linking experiments showing an increased specific incorporation of 125I-VIP in a major 66,000-dalton component during differentiation. A slight increase in receptor affinity is also observed during differentiation with Kd ranging from 0.39 nM at day 5 to 0.08 nM at day 30. These data indicate that one population of VIP receptors accumulates during Caco-2 cell differentiation, representing the crucial event in the development of adenylate cyclase response to the peptide.     

The Wilms' tumor suppressor Wt1 is associated with the differentiation of retinoblastoma cells.

We have demonstrated recently that Wilms' tumor suppressor 1 (Wt1),in addition to its role in genitourinary formation,is required for the differentiation of ganglion cells in the developing retina. Here we provide further evidence that Wt1 is associated with neuronal differentiation. Thus, the retinoblastoma-derived human cell line, Y-79, contained robust amounts of Wt1 mRNA and protein. Wt1 expression was down-regulated upon laminin-induced differentiation of Y-79 into neuron-like cells. Inhibition of Wt1 with antisense oligonucleotides dramatically reduced the capacity of undifferentiated Y-79 cells to undergo neuronal differentiation, whereas sense and missense oligonucleotides had no effect. Wt1 immunoreactivity was also detected in solid retinoblastomas, in which it resided mainly in areas with moderate proliferative activity. These findings suggest a role for Wt1 in the differentiation of retinoblastoma cells. Furthermore, Wt1 expression in retinoblastoma may reflect the potential of these tumors to initiate the early steps of neuronal differentiation.     

Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells.

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.     

Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function.

Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.     

Germ cells in Hydra oligactis males. II. Evidence for a subpopulation of interstitial stem cells whose differentiation is limited to sperm production

Animals containing germline-restricted interstitial cells were obtained by treating males from a clone of Hydra oligactis with hydroxyurea (HU) to lower the interstitial population to 1 or 2 cells per animal. A 3-day HU treatment produced animals whose interstitial cells did not form somatic cells, but did produce sperm. The isolation of these cells in HU-treated animals has lead us to propose that the interstitial cell population may contain subpopulations which possess different growth dynamics and developmental potentials. Through asexual propagation, we have cloned several animals containing only sperm precursor interstitial cells and have examined the growth and differentiation behavior of these cells in offspring propagated over a 2-year period. Evidence has been obtained which demonstrates (1) the extensive self-renewal capacity of the sperm precursor interstitial cells, and (2) the restricted differentiation capacity of these interstitial stem cells. Factors which affect cells entering and traversing the spermatogenic pathway are also presented.     

Hybrid type glycolipids (lacto-ganglio series) with a novel branched structure. Their presence in undifferentiated murine leukemia cells and their dependence on differentiation

The structures of these glycolipids are hybrids of the lacto and ganglio series, which are characterized by the presence of GlcNAc beta 1----3 and GalNAc beta 1----4 linked to the Gal residue of Gal beta 1----4Glc beta 1----1Cer. This new hybrid series can be designated as "lacto-ganglio series." These glycolipids are present in undifferentiated murine leukemia cells. Their concentration declines with differentiation and they are virtually absent in differentiated M1+ cells, suggesting that lacto-ganglio structures could be markers of undifferentiated, malignant myeloid cells.