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

The human colon carcinoma cell line HT-29 adapted to grow in absence of glucose exhibits a typical enterocytic differentiation. In contrast, cells grown in glucose always remain undifferentiated. To investigate whether differentiated HT-29 cells express a Na(+)-dependent sugar transporter, isotopic tracer flux measurements of a non-metabolizable sugar analogue methyl alpha-D-glucoside (AMG) were undertaken. AMG accumulation in confluent monolayer of differentiated HT-29 cells was inhibited by replacement of sodium, phlorizin, phloretin, and glucose. Kinetic studies demonstrate the presence of only one Na(+)-dependent phlorizin-sensitive sugar transporter in differentiated HT-29 cells. Undifferentiated HT-29 cells cultured in the presence of glucose did not show a Na(+)-dependent AMG accumulation. As previously demonstrated for other markers of the enterocytic differentiation, this transporter has a growth-related expression. Moreover, it shares similar properties with the Na(+)-dependent glucose transport in the human fetal small intestine and colon. To demonstrate that the expression of the Na(+)-dependent sugar cotransporter can be modulated by glucose, differentiated HT-29 cells grown in glucose-free medium were switched to 25 mM glucose. In that condition the Na(+)-dependent AMG uptake was almost abolished. However, when these cells were switched back to glucose-free medium, the Na(+)-dependent AMG uptake was restored, although at a lower level. These experiments show that differentiated HT-29 cells are a good cellular model to study the regulation of the Na(+)-dependent sugar transporter.

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

The Journal of Cell Biology ◽

10.1083/jcb.104.5.1199 ◽

1987 ◽

Vol 104 (5) ◽

pp. 1199-1205 ◽

Cited By ~ 62

Author(s):

G Trugnan ◽

M Rousset ◽

I Chantret ◽

A Barbat ◽

A Zweibaum

Keyword(s):

Complex Form ◽

Molecular Forms ◽

Posttranslational Processing ◽

Rapid Degradation ◽

Differentiated Cells ◽

Undifferentiated Cells ◽

High Mannose ◽

Complex Forms ◽

Ht 29 ◽

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.

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THE CELL LINES CACO-2, T84, AND HT-29: MODELS OF ENTEROCYTIC DIFFERENTIATION AND FUNCTION

Journal of Pediatric Gastroenterology and Nutrition ◽

10.1097/00005176-199704000-00103 ◽

1997 ◽

Vol 24 (4) ◽

pp. 473 ◽

Cited By ~ 2

Author(s):

G. Bolte ◽

K. Beuermann ◽

M. Stern

Keyword(s):

Cell Lines ◽

And Function ◽

Ht 29 ◽

Enterocytic Differentiation

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

Reproduction Nutrition Development ◽

10.1051/rnd:19900305 ◽

1990 ◽

Vol 30 (3) ◽

pp. 325-330 ◽

Cited By ~ 1

Author(s):

E. Ogier-Denis ◽

C. Bauvy ◽

M. Aubery ◽

P. Codogno ◽

C. Sapin ◽

...

Keyword(s):

Differentiation Process ◽

Early Marker ◽

Ht 29 ◽

Enterocytic Differentiation

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Lysosomal α-glucosidase: cell-specific processing and altered maturation in HT-29 colon cancer cells

Biochemical Journal ◽

10.1042/bj3140033 ◽

1996 ◽

Vol 314 (1) ◽

pp. 33-40 ◽

Cited By ~ 7

Author(s):

Clara FRANCÍ ◽

Gustavo EGEA ◽

Rosa ARRIBAS ◽

Arnold J. J. REUSER ◽

Francisco X. REAL

Keyword(s):

Colon Cancer ◽

Cancer Cells ◽

Protein Trafficking ◽

Cultured Cells ◽

Brefeldin A ◽

Colon Cancer Cells ◽

Intracellular Traffic ◽

Synthesis And Processing ◽

Golgi Network ◽

Ht 29

We have previously described the abnormal localization of resident Golgi proteins and O-glycans in the rough endoplasmic reticulum of mucin-secreting HT-29 M6 colon cancer cells, suggesting altered protein trafficking in these cells [Egea, Franci~A, Gambús, Lesuffleur, Zweibaum and Real (1993) J. Cell Sci. 105, 819–830]. In the present work, we have chosen lysosomal α-glucosidase as a reporter to examine the intracellular traffic of glycoproteins in M6 cells. We have compared the synthesis and processing of α-glucosidase in mucin-secreting M6 cells and in Caco-2 colon cancer cells, the latter resembling normal absorptive intestinal epithelium. Our results show that α-glucosidase processing and secretion is markedly delayed in M6 cells as compared with Caco-2 cells or normal fibroblasts, and this delay is caused by an accumulation of α-glucosidase precursor form in the trans-Golgi network. Furthermore, treatment of Caco-2 cells with brefeldin A led to changes in α-glucosidase maturation similar to those observed in untreated M6 cells. To determine whether altered processing occurs in other cultured cells, a panel of cancer cell lines and cultures from normal exocrine pancreas were examined. In pancreas-derived cultures, α-glucosidase showed a processing pattern different from that described until now. Only HT-29 cells and HT-29-derived subpopulations displayed a defect in α-glucosidase maturation. In conclusion, α-glucosidase processing is more diverse than has previously been described; this finding may have tissue-specific functional implications.

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