Colon Adenocarcinoma Cell Line HT-29 Research Articles

RhoA-dependent switch between alpha2beta1 and alpha3beta1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation.

Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an alpha2beta1/alpha3beta1 integrin switch, alpha3beta1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of alpha3beta1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin alpha3beta1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar alpha2beta1/alpha3beta1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed.

Overexpression of folate binding protein α is one of the mechanism explaining the adaptation of HT29 cells to high concentration of methotrexate

The human colon adenocarcinoma cell line HT29 can be adapted to 10 −7- 10 −4 M concentrations of methotrexate (MTX). Cells adapted to 10 −4 M MTX have an enterocyte-like phenotype with DHFR gene amplification. Presently, we hypothetized that an increased expression of folate binding protein (FBP) may participate to the MTX resistance of 10 −4 MTX HT29 cells. The cDNA FBPα/β-actin ratio of amplified transcripts was 4.8- and 1.5- fold higher in 10 −4 and in 10 −7 M MTX HT29 respectively, than in standard type HT29 cells. An increase of transcript level was observed when decreasing folic acid concentration. PI-PLC cleaved 7.7 times more membrane FBP in 10 −4 M than in 10 −7 M MTX and wild type HT29 cells. In contrast to 10 −7 M MTX cells, growth of 10 −4 M MTX cells was dependent on folic acid concentration and abolished at a concentration lower than 0.9 μM. In conclusion, the adaptive mechanism of HT29 cells resistant to 10 −4 M MTX is the result of the synergistic overexpression of both DHFR and FBPα. Overexpression of FBPα may be related to the enterocyte-like phenotype of the cells.

Butyrate and propionate downregulate ERK phosphorylation in HT-29 colon carcinoma cells prior to differentiation.

We have characterized the effects of different short-chain fatty acids (SCFAs) on cell growth and differentiation as well as the phosphorylation state of ERK1 and 2 in the human colon adenocarcinoma cell line HT-29. Of the five SCFAs tested, only butyrate and propionate impaired cellular proliferation. Moreover, butyrate and propionate specifically resulted in a decrease in ERK1 and 2 phosphorylation at 3 and 6 hours post-treatment, suggesting a correlation between the ability of these SCFAs to inhibit cellular proliferation and decrease ERK phosphorylation. Notably, the decrease in ERK phosphorylation was observed prior to the induction of the differentiation markers alkaline phosphatase (AP) and carcinoembryonic antigen (CEA) by butyrate and propionate from days 6 to 18 post-treatment. In the case of butyrate- and propionate-induced differentiation, ERK phosphorylation is a marker and may play a role in the proliferation and/or differentiation states of this cell line.

Effect of eicosapentaenoic acid on the proliferation and incidence of apoptosis in the colorectal cell line HT29.

Fish oil has been shown to reduce the induction of colorectal cancer in animal models by a mechanism which may involve suppression of mitosis, increased apoptosis, or both. We used the human colonic adenocarcinoma cell line HT29 to explore the effects of the long-chain n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) on cell proliferation and death in vitro. Cells were cultured in media containing EPA at 5, 10, and 15 microg/mL. Cell number and thymidine incorporation were used to quantify proliferation, and cell cycle effects were studied using flow cytometry. Gel electrophoresis, annexin-V binding, and morphological criteria were used to characterize apoptosis. Adherent cells and freely floating detached cells were treated as two distinct populations. In the presence of EPA at 10 and 15 microg/mL there was a marked reduction in the growth rate of adherent HT29 colonies, owing to an increased detachment of adherent cells. After treatment with 10 or 15 microg/mL EPA the proportion of adherent cells in S-phase increased, indicating either a block in late S-phase or early G2. Floating cells showed evidence of extensive DNA cleavage, but the proportion of floating cells with sub GO DNA content declined on treatment with 10 or 15 microg/mL EPA even though the number of floating cells increased. We conclude that EPA does not inhibit mitosis of adherent cells, but increases the rate at which they become detached from the substrate, probably at an early stage in the initiation of apoptosis. This mechanism may be analogous to "anoikis," or induction of apoptosis in response to loss of cell contact, and may contribute to the anticarcinogenic effects of fish oil in vivo.

A diet high in fat and meat but low in dietary fibre increases the genotoxic potential of 'faecal water'.

To determine the effects of different diets on the genotoxicity of human faecal water, a diet rich in fat, meat and sugar but poor in vegetables and free of wholemeal products (diet 1) was consumed by seven healthy volunteers over a period of 12 days. One week after the end of this period, the volunteers started to consume a diet enriched with vegetables and wholemeal products but poor in fat and meat (diet 2) over a second period of 12 days. The genotoxic effect of faecal waters obtained after both diets was assessed with the single cell gel electrophoresis (Comet assay) using the human colon adenocarcinoma cell line HT29 clone 19a as a target. The fluorescence and length of the tails of the comet images reflects the degree of DNA damage in single cells. The mean DNA damage, expressed as the ratio of tail intensity (fluorescence in the tail) to total intensity of the comet after incubation with faecal water from volunteers consuming diet 1 was about twice as high as for diet 2. The susceptibility of the cells incubated with faecal water to DNA damage caused by additional hydrogen peroxide treatment showed no significant differences between the two diets. Generation of oxidized pyrimidine and purine bases revealed no differences after pretreatment with both types of faecal water. The results indicate that diets high in fat and meat but low in dietary fibre increase the genotoxicity of faecal water to colonic cells and may contribute to an enhanced risk of colorectal cancer.

DOTA-lanreotide: a novel somatostatin analog for tumor diagnosis and therapy.

Long acting somatostatin-14 (SST) analogs are used clinically to inhibit tumor growth and proliferation of various tumor types via binding to specific receptors (R). We have developed a 111In-/90Y-labeled SST analog, DOTA-(D)betaNal1-lanreotide (DOTALAN), for tumor diagnosis and therapy. 111In-/90Y-DOTALAN bound with high affinity (dissociation constant, Kd, 1-12 nM) to a number of primary human tumors (n = 31) such as intestinal adenocarcinoma (n = 17; 150-4000 fmol/mg protein) or breast cancer (n = 4; 250-9000 fmol/mg protein). 111In-/90Y-DOTALAN exhibited a similar high binding affinity (Kd, 1-15 nM) for the human breast cancer cell lines T47D and ZR75-1, the prostate cancer cell lines PC3 and DU145, the colonic adenocarcinoma cell line HT29, the pancreatic adenocarcinoma cell line PANC1, and the melanoma cell line 518A2. When expressed in COS7 cells, 111In-DOTALAN bound with high affinity to hsst2 (Kd, 4.3 nM), hsst3 (Kd, 5.1 nM), hsst4 (Kd, 3.8 nM), and hsst5 (Kd, 10 nM) and with lower affinity to hsst1 (Kd, approximately 200 nM). The rank order of displacement of [125I]Tyr11-SST binding to hsst1 was: SST (IC50, 0.5 nM) >> DOTALAN (IC50, 154 nM) > lanreotide (LAN) approximate to Tyr3-octreotide (TOCT) approximate to DOTA-Tyr3-octreotide (DOTATOCT) approximate to DOTA-vapreotide (DOTAVAP; IC50, >1000 nM); that to hsst2 was: DOTATOCT approximate to TOCT approximate to DOTALAN approximate to SST approximately LAN approximate to DOTAVAP (IC50, 1.4 nM); that to hsst3 was: SST (IC50, 1.2 nM) > DOTALAN = LAN (IC50, 15 nM) approximate to TOCT (IC50, 20 nM) approximate to DOTAVAP (IC50, 28 nM) > DOTATOCT (IC50, 73 nM); that to hsst4 was: SST (IC50, 1.8 nM) approximate to DOTALAN (IC50, 2.5 nM) > LAN (IC50, 22 nM) >> DOTATOCT approximate to DOTAVAP approximate to TOCT (IC50, >500 nM); and that to hsst5 was: DOTALAN (IC50, 0.45 nM) > SST (IC50, 0.9 nM) > TOCT (IC50, 1.5 nM) > DOTAVAP (IC50, 5.4 nM) >> LAN (IC50, 21 nM) > DOTATOCT (IC50 260 nM). In Sprague Dawley rats (n = 10), 90Y-DOTALAN was rapidly cleared from the circulation and concentrated in hsst-positive tissues such as pancreas or pituitary. Taken together, our results indicate that 111In-/90Y-DOTALAN binds to a broad range of primary human tumors and tumor cell lines, probably via binding to hsst2-5. We conclude that this radiolabeled peptide can be used for hsst-mediated diagnosis (111In-DOTALAN) as well as systemic radiotherapy (90Y-DOTALAN) of human tumors.

Induced differentiation in HT29, a human colon adenocarcinoma cell line.

The human colon adenocarcinoma cell line HT29 displays an undifferentiated phenotype under standard growth conditions. When these cells were cultured for 21 days and then treated with forskolin, most of the cells formed brush borders on their apical surfaces. Brush border formation was inhibited by cytochalasin D but not by colchicine. Colchicine, nocodazole and taxol were found to induce differentiation and apoptosis in HT29 cells. Differentiation was characterized by flattening of the cells, formation of brush borders on apical surfaces and tight junctions between adjacent cells. Apoptosis was characterized by detachment of round cells from the cell layer, condensation of nuclear DNA and annexin V binding to cell surfaces. Treatment with colchicine or forskolin induced the association of E-cadherin to the cytoskeleton fraction of subconfluent HT29 cells. This effect was less prominent in post confluent cells. Our data indicate that microtubule-interfering agents may serve as an important tool in the study of differentiation and apoptosis in intestinal carcinoma.

The three-dimensional structure of human RNase 4, unliganded and complexed with d(up), reveals the basis for its uridine selectivity

The RNase 4 family is unique among RNase enzymes, displaying the highest level of sequence similarity and encompassing the shortest polypeptide chain. It is the only one showing high specificity. The human representative is an intracellular and plasma enzyme, first isolated from colon adenocarcinoma cell line HT-29. The crystal structures of human recombinant RNase 4, unliganded and in complex with d(Up), have been determined, revealing in the unique active site an explanation for the uridine specificity. Arg101, at a position not involved in catalysis in the other RNase enzymes, penetrates the enzyme moiety shaping the recognition pocket, a flip that is mediated by the interaction with the (shorter chain) C-terminal carboxylate group, providing an anchoring point for the O4 atom of the substrate uridine. The bulky Phe42 side-chain forces Asp80 to be in the χ1 = −72.49° rotamer, accepting a hydrogen bond from Thr44, further converting the latter into a hydrogen bond acceptor. This favours an interaction with the −NH-donor group of uridine at position 3 over that with the =N-acceptor of cytidine. The two chemical groups that distinguish uracyl from cytosine are used by the enzyme to discriminate between these two bases.

Interferon-γ-Induced Differentiation and Apoptosis of HT29 Cells: Dissociation of (Glucosyl)ceramide Signaling

Recently, (glyco)sphingolipids (SL) like ceramide (Cer) and glucosylceramide (GlcCer) have been shown to be involved in signaling pathways leading to differentiation and apoptosis in several cell types, including the colon adenocarcinoma cell line HT29. Intracellular levels of Cer can be modulated by ligands such as interferon-γ (IFNγ). In the present study we show that IFNγ, depending on its concentration, has both differentiation- and apoptosis-inducing effects on HT29 cells. Since both phenomena have been related to SL-mediated signaling in other cell types, we next examined whether IFNγ was able to induce changes in the SL levels of HT29 cells. Remarkably, no significant changes in these levels could be revealed, implying that SL are not involved in IFNγ-induced differentiation and/or apoptosis of HT29 cells. This observation provides evidence for the hypothesis that SL-mediated signaling pathways might be more cell type specific than is generally assumed.

Down-regulation of Vascular Endothelial Growth Factor in a Human Colon Carcinoma Cell Line Transfected with an Antisense Expression Vector Specific for c-src

Vascular endothelial growth factor (VEGF) is implicated in the angiogenesis of human colon cancer. Recent evidence suggests that factors that regulate VEGF expression may partially depend on c-src-mediated signal transduction pathways. The tyrosine kinase activity of Src is activated in most colon tumors and cell lines. We established stable subclones of the human colon adenocarcinoma cell line HT29 in which Src expression and activity are decreased specifically as a result of a transfected antisense expression vector. This study determined whether VEGF expression is decreased in these cell lines and whether the smaller size and reduced growth rate of antisense vector-transfected cell lines in vivo might result, in part, from reduced vascularization of tumors. Northern blot analysis of these cell lines revealed that VEGF mRNA expression was decreased in proportion to the decrease in Src kinase activity. Under hypoxic conditions, cells with decreased Src activity had a <2-fold increase in VEGF expression, whereas parental cells had a >50-fold increase. VEGF protein in the supernatants of cells was also reduced in antisense transfectants compared with that from parental cells. In nude mice, subcutaneous tumors from antisense transfectants showed a significant reduction in vascularity. These results suggest that Src activity regulates the expression of VEGF in colon tumor cells.

Production of arachidonic acid metabolites by the colon adenocarcinoma cell line HT29 cl.19A and their effect on chloride secretion.

Eicosanoids were found in large amounts in the colonic mucosa of patients suffering from inflammatory bowel diseases and colonic adenocarcinoma. The aim of this study was to evaluate the role of the intestinal epithelial cells in the arachidonic acid metabolism and their functional response to certain eicosanoids. We used the human adenocarcinoma epithelial cell line HT29 cl.19A cell, which is an in vitro model of colon carcinoma and ion transport. These cells were found to express 5- and 15-lipoxygenase, leukotriene A4 hydrolase and cyclooxygenase-1 and -2 mRNAs. We observed an arachidonic acid metabolism via 5-lipoxygenase pathway despite the lack of FLAP mRNA expression and that certain eicosanoids such as hydroperoxy- and hydroxyeicosatetraenoic acids stimulate chloride secretion.

Interferon-γ Modulates a p53-independent Apoptotic Pathway and Apoptosis-related Gene Expression

Interferon (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p53 mutants, to tumor necrosis factor-alpha-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p53 function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP32 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-3 (ICE-LAP3, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p53. The IFN-responsive transcriptional activator interferon regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p53-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.

Analysis of Apoptosis by Confocal Laser Scanning Microscopy based on Three-dimensional Images.

The present study examined the changes of karyomorphism in cultured cells (human colon adenocarcinoma cell line HT-29 and human oral squamous cell carcinoma cell line KB) using a confocal laser scanning microscope (ACAS 570 laser cytometer). After INF-γ stimulation, Apoptosis of HT-29 cells and KB cells were induced by the Fas/Fas ligand system and cychroheximide, respectively. Both types of apoptosis-induced cells were stained with propidium iodide (PI) and distribution of nuclear DNA content was determined, and the three-dimensional image was analyzed. The results were as follows. Simultaneous visualization of quantitative and morphological changes of DNA, using PI fluorescence as an indicator, clarified that the distribution of DNA content in the nucleus was remarkably uneven; it was low in the central area and high in the marginal area. In addition, three-dimensional images revealed a concave area in the center of the cell nuciei, fragmented irregular nuclei, and a doughnut-shaped and hemispheric form. Thus, the three-dimensional study presented a clear picture of the nuclear structures which characteristically appear in cells during the process of apoptosis. Confocal laser scanning microscopy was confimed as useful in the observation of apoptosis.

Change of calretinin expression in the human colon adenocarcinoma cell line HT29 after differentiation

Calretinin is a Ca 2+-binding protein of the EF-hand family which is expressed in colon adenocarcinomas and colon-derived tumor cell lines (e.g. WiDr), but is absent from normal human enterocytes. Its function has not as yet been elucidated, but some lines of evidence lead us to postulate its involvement in cell proliferation in these cells. In order to test whether calretinin is correlated with an undifferentiated, proliferating, or with a differentiated, state of cells, its expression was studied in the human colon adenocarcinoma clonal cell line HT29-18, which can be caused to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (glucose starvation differentiation). Treatment of HT29-18 cells with galactose led to a drop in the calretinin mRNA level and in protein expression as evidenced by immunocytochemical staining and Western blot analysis of cytosolic cell extracts. These results suggest that calretinin is present in HT29-18 cancer cells, mostly in those which are in the undifferentiated state. The possibility that calretinin is involved in maintaining the cells in an undifferentiated (cancerous) state is discussed.

Characterisation of the complement-regulatory proteins decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46) on a human colonic adenocarcinoma cell line.

To avoid destruction by complement, normal and malignant cells express membrane glycoproteins that restrict complement activity. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and protectin (CD59), which are all expressed on colonic adenocarcinoma cells in situ. In this study we have characterised the C3/C5 convertase regulators DAF and MCP on the human colonic adenocarcinoma cell line HT29. DAF was found to be a glycosyl-phosphatidylinositol-anchored 70-kDa glycoprotein. Blocking experiments with F(ab')2 fragments of the anti-DAF monoclonal antibody BRIC 216 showed that DAF modulates the degree of C3 deposition and mediates resistance to complement-mediated killing of the cells. The expression and function of DAF were enhanced by tumour necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta). Cells incubated with interferon gamma (IFN gamma) did not alter their DAF expression. Two MCP forms were expressed, with molecular masses of approximately 58 kDa and 68 kDa, the lower form predominating. MCP expression was up-regulated by IL-1 beta, but not by TNF alpha or INF gamma. Expression of DAF and MCP promotes resistance of colonic adenocarcinoma cells to complement-mediated damage, and represents a possible mechanism of tumour escape.

Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon gamma.

Coeliac disease is precipitated in susceptible subjects by ingestion of wheat gluten or gluten related prolamins from some other cereals. The disease is strongly associated with certain HLA-DQ heterodimers, for example, DQ2 (DQ alpha 1*0501, beta 1*0201) in most patients and apparently DQ8 (DQ alpha 1*0301, beta 1*0302) in a small subset. Gluten specific T cell clones (TCC) from coeliac intestinal lesions were recently established and found to be mainly restricted by HLA-DQ2 or HLA-DQ8. Antigen induced production of cytokines was studied in 15 TCC from three patients, 10 being DQ2 and five DQ8 restricted. Cell culture supernatants were prepared by stimulation with gluten peptides in the presence of DQ2+ or DQ8+ Epstein-Barr virus transformed B cells as antigen presenting cells (APC). Supernatants were analysed for cytokines by bioassays, ELISA, and CELISA. Cellular cytokine mRNA was analysed semi-quantitatively by slot blotting and polymerase chain reaction (PCR). All TCC were found to secrete interferon (IFN) gamma, often at high concentrations (> 2000 U/ml); some secreted in addition interleukin (IL) 4, IL 5, IL 6, IL 10, tumour necrosis factor (TNF), and transforming growth factor (TGF) beta. The last TCC thus displayed a Th0-like cytokine pattern. However, other TCC produced IFN gamma and TNF but no IL 4, or IL 5, compatible with a Th1-like pattern. In conclusion, most DQ8 restricted TCC seemed to fit with a Th0 profile whereas the DQ2 restricted TCC secreted cytokines more compatible with a Th1 pattern. The TCC supernatants induced upregulation of HLA-DR and secretory component (poly-Ig receptor) in the colonic adenocarcinoma cell line HT-29.E10, most probably reflecting mainly the high IFN gamma concentrations. This cytokine, particularly in combination with TNF alpha, might be involved in several pathological features of the coeliac lesion. The characterised cytokine profiles thus support the notion that mucosal T cells activated in situ by gluten in a DQ restricted fashion play a central part in the pathogenesis of coeliac disease.

Selected eicosanoids increase the proliferation rate of human colon carcinoma cell lines and mouse colonocytes in vivo

Eicosanoids have been implicated in colon carcinogenesis, but their role remains unclear. The levels of PGE 2 are elevated in colon cancer tissues and in blood draining colon tumors. The effect of eicosanoids on the proliferation of colonic cells is unknown. We studied the effect of several prostaglandins (PGs) and leukotriene (LT)B 4 on the proliferation rate of the human colon adenocarcinoma cell lines SW1116 and HT-29 and of 16,16-dimethyl PGE 2 (dmPGE 2) on the colon of BALB/c mine. PGs E 2, F 2α, I 2, the methyl ester of, PGE 2, dmPGE 2, and LTB 4 (10 −10, 10 −8, 10 −6 M), administered for up to 72 h, stimulated cell proliferation in SW1116 cells and all t but PGF 2α and PGI 2 stimulated proliferation in HT-29 cells. The proliferative effect was time- and concentration-dependent. However, in SW1116 cells the response to PGs was ‘bell-shaped’, being maximal at 10 −8 M, with the 10 −10 and 10 −6 M concentrations being less effective. In HT-29 cells, the addition of methyl groups to the PGE 2 molecule increased the proliferative affect. None of these eicosanoids affected the distribution of these cells in the cell cycle or their rate of programmed cell death (apoptosis). dmPGE 2 stimulated 3.6-fold the proliferation of colonocytes in normal BALB/c mice. This was determined by bivariate flow cytometric analysis of the expression of proliferating cell nuclear antigen (PCNA) in virtually pure populations of mouse colonocytes. dmPGE 2 did not alter the cell cycle distribution of these cells. We conclude that several PGs as well as LTB 4 stimulate the proliferation of human colon carcinoma cells in vitro, while dmPGE 2 has a similar effect on mouse colonocytes in vivo. These findings raise the possibility that eicosanoids may contribute to colonic carcinogenesis by stimulating the proliferation rate of tumor cells in the colon.

Differentiation-dependent autophagy controls the fate of newly synthesized N-linked glycoproteins in the colon adenocarcinoma HT-29 cell line

Our previous results have demonstrated that, in undifferentiated human colon cancer HT-29 cells, a pool of glycoproteins bearing high-mannose oligosaccharides rapidly escapes the exocytic pathway to be degraded in the lysosomal compartment [Trugnan, Ogier-Denis, Sapin, Darmoul, Bauvy, Aubery and Codogno (1991) J. Biol. Chem. 266, 20849-20855]. We report here on the mechanism that governs this degradative pathway. Using pulse-chase experiments in combination with subcellular fractionation, we have observed that the sequestration of high-mannose glycoproteins in lysosomes was impaired by drugs which interfere with the autophagic-lysosomal pathway. The accumulation of high-mannose glycoproteins in the lysosomal fraction was shown to be part of the general autophagic pathway constitutively expressed in undifferentiated cells, as independently measured by the sequestration of the cytosolic enzyme lactate dehydrogenase and electroloaded raffinose. Furthermore, when HT-29 cells were cultured under differentiation-permissive conditions, the decreased accumulation of high-mannose glycoproteins in the lysosomal compartment was correlated with the decrease in autophagy.

The Influence of Tumour Necrosis Factor‐γ, Interleukin‐1β and Interferon‐γ on the Expression and Function of the Complement Regulatory Protein CD59 on the Human Colonic Adenocarcinoma Cell Line HT29

CD59 is a 18-25 kDa glycoprotein which, by inhibiting the formation of the membrane attack complex, protects homologous cells from complement mediated damage. We have described recently the expression and complement regulatory function of CD59 on colonic adenocarcinoma cells both in vivo and in vitro. In this study we have examined the influence of cytokines on the expression and complement regulatory function of CD59 on the colonic adenocarcinoma cell line HT29. CD59 expression on the HT29 cells was up-regulated after stimulation by mononuclear cells activated by mixed lymphocyte reaction and by culture supernatants from activated mononuclear cells. Similarly, a dose-dependent increase in CD59 expression was observed after stimulation with both tumour necrosis factor-alpha and interleukin-1 beta. A dose-dependent increase in the level of CD59 expression was also seen using low concentrations of interferon-gamma (IFN-gamma), while CD59 expression on cells cultured with high IFN-gamma concentrations was comparable to non-stimulated cells. Cytokine treated cells were more resistant to lysis by homologous complement than non-stimulated cells, and the increase in CD59 expression was shown to be partially responsible for this. The present data strengthen the role of CD59 as a possible participant in tumour escape.

Expression of Hormone-Sensitive Lipase in the Human Colon Adenocarcinoma Cell Line HT29

Hormone-sensitive lipase expression was studied in the human colon adenocarcinoma cell line, HT29. Diacylglycerol lipase and cholesterol oleate activities in HT29 cells were inhibited by known inhibitors of hormone-sensitive lipase (diethyl- p-nitrophenyl phosphate, NaF and HgCl 2) to the same extent as in human adipocytes. A polyclonal antiserum directed against rat hormone-sensitive lipase inhibited 89 % of HT29 cell lipase activity. HT29 hormone-sensitive lipase was the same size as the adipocyte enzyme as was its mRNA. Complete homology between mRNA sequences in HT29 and adipocyte was demonstrated using ribonuclease protection assay, These data are consistent with the expression of a protein closely related, if not identical, to the enzyme expressed in human adipose tissue. HT29 is the first human cell line where hormone-sensitive lipase expression has been shown.