HeLa cells butyrate-treated

Butyrate treated Hela cells (2) and KB cells showed marked increases in the amounts of G 3 gangliosides and elevated levels of the enzyme, CMP sialic acid lactosylceramide sialosyltrans-ferase, required for its synthesis. 

Morphological changes were prevented in butyrate-treated HeLa cells by actinomycin D and cycloheximide (2,8,13). After removal of butyrate, the cells reverted to a normal morphology over a 24 h time course (2,8,12,13). When butyrate-treated cells were detached from the cuTture dishes with trypsin, they assumed a spherical shape and, when replated in the absence of butyrate, their neurite-like processes transiently re-extended (13). This re-extension was blocked when cycloheximide but not the calcium ionophore was included during the initial exposure of the cells to butyrate (13). Process formation, however, did resume in the presence of cycloheximide (1 3). These results were interpreted as indicating that the fatty acid induces a protein(s) required for process formation which can accumulate in the absence of processing and promote processing in the absence of inducer (13). 

Figure 1. Scanning electron micrographs of untreated (top) and butyrate-treated HeLa cells (bottom). Photographs taken by Saleem Jahangeer at George Washington University (/.J280).

Figure 5. Effect of labeled and unlabeled choleragen concentrations on n5I-choleragen binding to control and butyrate-treated HeLa cells...

Gangliosides from butyrate-treated HeLa Cells ... 

The above results indicate that the increased choleragen binding to butyrate-treated HeLa cells is associated with increased GMl content. This was confirmed with Friend erythroleu-kemic cells (Table VII). Untreated Friend cells have measurable... 

Increased GM3 content was also observed in another strain of HeLa exposed to butyrate but not in butyrate-treated normal human fibroblasts (experiments in collaboration with E. Stanbridge, University of California at Irvine and R. 0. Brady, NINCDS). Butyrate appeared to have similar effects on GM3 biosynthesis in KB cells, another human carcinoma-derived cell line (20). Butyrate-treated KB cells had 9-fold elevated levels of sialyl transferase activity. In contrast, butyrate as well as dibutyryl-... 

HeLa cells were treated with 5 mM butyrate for 48 h or with 1 yM GM1 for 1 h. Both types of treated cells bound similar amounts of 125I-choleragen and about 60-fold more than did control cells (Fig. 6a). When the cells were exposed to a saturating dose of toxin, the time course and extent of cyclic AMP accumulation were similar for both the butyrate-treated and the GM1-treated HeLa cells and more rapid than in the control cells (Fig. 6b). Thus, butyrate induces choleragen receptors in HeLa cells that are functionally equivalent to those created by incorporation of exogenous GM1. 

When HeLa cells were.cultured in medium supplemented with 5 mM sodium butyrate, their content of GM3 increased (Fig.2a). Increases varied from 3.5 to 5-fold depending on the experiment (4,8,12,13). When the butyrate was removed and the cells were cultured in normal medium for 24 h, the GM3 levels returned to those found in untreated cells (Fig. 2a). Similar results were observed when N-[acetyl-3H]-D-mannosamine, a precursor of sialic acid, was also included in the culture medium. In the butyrate-treated cells, radioactivity associated with GM3 increased 6.5-fold and 24 h after butyrate was removed, the amount of labeling returned to control values (Fig. 2b). We also were able to label the GM3 by means of a cell surface labeling technique. Control and butyrate-treated cells were exposed to 10 mM sodium periodate and the oxidized sialyl residues were reduced with NaBSfy. There was 5.5-fold more 3h associated with the GM3 recovered from the butyrate-... 

HeLa cells were cultured in medium containing N[acetyl-3H]-D-mannosamine (50 pCit mL) for 24 hr with (solid bars) and without (open bars) 5 mM sodium butyrate. In addition, butyrate-treated cells were cultured an additional 24 hr in fresh medium (without label and butyrate) (hatched bars). The cells were harvested and analyzed for GM3 content and radioactivity. (Data from Refs. 8, Vi.)... 

Exposure of HeLa cells to butyrate had no effect on the activity of GM3-sialidase when GM3 specifically labeled in the sialic acid residue was used as substrate (Fig. 3a). We were unable to detect any "ecto"-sialidase activity in either control or butyrate-treated cells (14) although others have postulated that such an enzyme is important in regulating plasma membrane gangliosides (15,16). In contrast, the activity of the specific sialyl transferase involved in GM3 biosynthesis increased over 20-fold following butyrate treatment (Fig. 3b). The effect was specific as activities of the other glycosphingolipid transferases that could be measured in HeLa cells were not altered in butyrate-treated cells (4,8,17). 

If GM1 is the choleragen receptor, then butyrate-treated cells should have an increase in GM1 content. This is demonstrated in Table IV. Although GM1 could not be detected in control HeLa cells, they would contain less than 1 pmol per mg protein based on the limits of the sensitivity of the analytical procedure (5). GM1 was quantitated in the butyrate-treated cells (28.5 pmol per mg protein) and this increase is similar to the 32-fold increase in toxin binding observed in cells from the same experiment. The delipidated residue contained less than 1% of the toxin binding found in intact cells (Table IV). In addition, removal of the cells from the culture dishes with trypsin as opposed to the mechanical scraping routinely used had no effect on 125I-cholera-gen binding to either control or butyrate-treated cells (5). 

The formation of the neurite-like processes appears to be dependent on assembly of microtubules as colchicine and Colcemid, antimicrotubule drugs, prevented shape changes in the presence of butyrate (2,8). The amount of tubulin per cell did not change when HeLa were treated with butyrate (R.C.Henneberry, unpublished observations). The role of microtubule assembly was further explored with a calcium ionophore which alters intracellular calcium levels and thus promotes microtubule depolymerization. 

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