Recent results have shown that autophagic sequestration in the human colon cancer cell line HT-29 is controlled by the pertussis toxin-sensitive heterotrimeric G i3 protein. Here we show that transfection of an antisense oligodeoxynucleotide to the ␣ i3 -subunit markedly inhibits autophagic sequestration, whereas transfection of an antisense oligodeoxynucleotide to the ␣ i2 -subunit does not change the rate of autophagy in HT-29 cells. Autophagic sequestration was arrested in cells transfected with a mutant of the ␣ i3 -subunit (Q204L) that is restricted to the GTP-bound form. In Q204L-expressing cells, 3-methyladenine-sensitive degradation of long lived [14 C]valine-labeled proteins was severely impaired and could not be stimulated by nutrient deprivation. Autophagy was also reduced when dissociation of the ␤␥ dimer from the GTP-bound ␣ i3 -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 ␣ i3 -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 ␣ i3 -subunit (C351S) or S47N ␣ i3 -subunit (S47N/C351S) exhibit a high rate of autophagy.

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.

Epidemiological and in vivo and in vitro experimental studies have suggested that fermented milks may interfere with the emergence and/or the development of colon cancer. The results, however, remain inconclusive. This prompted us to develop a new approach based on the use of HT-29, a cultured human colon cancer cell line, to study at the cellular level the effect of fermented milks on colon cancer cell growth and differentiation characteristics. Undifferentiated HT-29 cells have been grown in the continuous presence of milks fermented by one of the following bacterial populations: Lactobacillus helveticus, Bifidobacterium, L.acidophilus or a mix of Streptococcus thermophilus and L. bulgaricus. Penicillin G was added to the cell culture medium, resulting in a complete blockade of bacterial growth without significant effect on bacterial viability. One out of the four bacteria species studied, namely L.acidophilus, was without effect on both cell growth and differentiation. The three other bacterial strains induced a significant, although variable, reduction in the growth rate of HT-29 cells, which resulted in a 10-50% decrease in the cell number at steady-state (i.e. at cell confluency). The most efficient strains in lowering the HT-29 growth rate were L. helveticus and Bifidobacterium. Concomitantly, the specific activities of dipeptidyl peptidase IV (DPP IV), a sensitive and specific marker of HT-29 cell differentiation, and that of three other brush border enzymes (sucrase, aminopeptidase N and alkaline phosphatase) were significantly increased, thus suggesting that these cells may have entered a differentiation process. Altogether, these results indicate that the use of cultured colon cancer cells may be a useful tool to further study the effect of fermented milks on colon cancer and that bacterial strains may exert a different and specific effect on cancer cell growth and differentiation when used in fermented milk products.

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After treatment with swainsonine, an inhbitor of both lysosomal a-mannosidase and Golgi a-mannosidase-I1 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 a-mannosidase-I I activity in both cell populations. Pulse/chase experiments carried out in the presence of swainsonine revealed that the persistence of high-mannosetype 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 a-mannosidase(s), whereas in differentiated HT-29 cells glycoproteins enter a compartment in which a-mannosidase 11 (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.Investigation of glycoprotein metabolism and particularly of mannosidase functions has been facilitated by the availability of the indolizidine alkaloid, swainsonine [l], a potent inhibitor of liver lysosomal mannosidase [2, 31 and Golgi amannosidase 11 [3,4]. The latter enzyme, located in the medial part of the Golgi apparatus [3 -71, catalyzes the final hydrolytic step in the pathway for the biosynthesis of glycoproteins bearing complex-type N-glycans, namely, the removal of a(1-3)-linked and a(1-6)-linked mannosyl groups from GlcNAc-Man5-GlcNAc2 to yield GlcNAc-Man3-GlcNAc2 [8 -101. Blocking this maturation by swainsonine has been shown to result in the production of hybrid-type oligosaccharides instead of complex-type N-glycans [4, 11 -131. Furthermore, it has been postulated that a-mannosidase, together with other hydrolases, plays a central role in the lysosomal catabolism of glycoproteins containing asparagine-linked oligosaccharides [4]. This supposition is based on the fact that oligosaccharides accumulate in the lysosomes of animals and humans with an inherited a-rnannosidase deficiency [l, 2, 141. Moreover, the ingestion of swainsonine-containing plants produces the same symptoms in animals as those observed in the lysosomal storage disease, a-mannosidosis [2, 41. Thus, this inhibitor may be a useful tool for studies of protein post-translational modification and intracellular traffic of glycoproteins.We have recently shown that N-glycan trimming is strongly impaired as a function of the state of enterocytic differentiation of HT-29 cells [15 -171. Indeed, the classical pattern of asparagine-linked oligosaccharide processing was observed in differentiated HT-29 cells, ...