Metabolism of low‐density lipoprotein in differentiated and undifferentiated HT29 colon cancer cells

Abstract

The metabolism of human low-density lipoproteins was studied in 2 subpopulations deriving from cells of HT29, a human colon carcinoma cell line. When grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G+ cells). From this heterogeneous population, a subpopulation with features of differentiated small-intestinal cells was selected by glucose deprivation (G- cells). The characteristics of the LDL receptor were first investigated. The results showed that the binding of 125I-LDL to G+ and G- cells performed at 4 degrees C was saturable and specific. The Kd values were not statistically different in the 2 cell subpopulations. The Bmax of G+ cells was 55 +/- 6 ng 125I-LDL/mg cell protein and showed no changes whatever the phase of culture. In G- cells, the Bmax was higher during the exponential phase of culture and decreased in the post-confluent phase (82 +/- 5 versus 15 +/- 6.8 ng 125I-LDL/mg cell protein). Cellular degradation of 125I-LDL was effective in both cell subpopulations but time-course studies showed that, in post-confluent G- cells, degradation was slowed as compared to G+ cells (4 hr vs. 2 hr to reach maximal degradation). The rate of LDL processing at 37 degrees C was enhanced by pre-incubation with FCS-supplemented medium, suggesting the existence of a serum component which stimulates the total degradation of 125I-LDL. Concerning regulation of the LDL receptor activity, we demonstrated that pre-incubation of G+ cells with LDL induced 80% down-regulation of receptor number in both phases of culture. This was also observed in G- cells during the exponential phase while only a 20% decrease of the receptor number was observed in post-confluent G- cells. The LDL degradation of G+ cells resulted in an inhibition of the cholesterogenic activity by 30% and 60% depending on the phase of culture. In G- cells, LDL pre-incubation inhibited cholesterol synthesis to the same extent (45%) in the exponential phase but did not affect the rate of cholesterol synthesis when cells were confluent. The defective regulatory role of LDL on receptor number and cholesterol synthesis suggests that, in the post-confluent differentiated cells, cholesterol derived from LDL does not reach the regulatory pool. Taken together, our findings indicate the existence of functional LDL receptors in the HT29 cell line, either in the differentiated or in the undifferentiated form.

Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines

“…Evidence exists for phenotypic change in terms of cholesterol metabolism with changing growth and differentiation states in colonic adenoCA cells. The observed inability of LDL pretreatment to reduce cholesterol synthesis in four colonic ade-noCA cell lines cultured at normal density is similar to observations reported by Villiard et al (58) for HT29 cells in stationary vs. exponential phase. This group also found LDLR activity to be more responsive vs. control under exponential (approximately 85%) than stationary (10%) conditions with a dramatic difference in the differentiated "G-" phenotype (58).…”

“…Our results demonstrate that in contrast to the fibroblast, colonic cells exhibited no downregulation of cholesterol synthesis by LDL at subconfluency (Table l). This variation of cellular density with lipoprotein metabolism also has been shown by many others in intestinal cells in culture (28,40,41) and in neoplastic cells (56,57).…”

“…Cholesterol dynamics for a given tumor cell reflect its current state of (i) differentiation which includes not only "poorly vs. well" differentiated characteristics but also the differences between large vs. small bowel phenotypes, and (ii) cellular density in monolayer culture which may or may not affect such differentiation. The colonic adenoCA cell lines HT-29 and CACO 2 have shown altered regulation of cholesterol metabolism with the state of differentiation (colonic vs. small intestinal) and confluency (28,41,42). When the adenoCA subline HT-29G is undifferentiated, acyl cholesterol acyl transferase (ACAT) activity was found to be absent (43).…”

“…Normal human (48) and murine (49) colonic epithelium have been shown to be LDLR positive by immunohistochemistry. Expression of the LDLR was found to vary in different portions of the small and large intestine (37,48,49), which may be imposed by the current state of differentiation (40), and growth (41,50,51) of these cells. In fact, immunohistochemical studies from this laboratory have shown that LDLR expression in human normal and neoplastic colon increases with the degree of differentiation of the tissue (48).…”

Low density lipoprotein and liposome mediated uptake and cytotoxic effect of N4-octadecyl-1-β-D-arabinofuranosylcytosine in Daudi lymphoma cells

“…LDL was obtained from fresh human plasma by sequential flotation ultracentrifugation (Viallard et al, 1990). Isolated LDL fractions were run on Sephadex G-25 columns (16 × 60 mm, Pharmacia, Uppsala, Sweden) for buffer exchange.…”