11

Biochimica et Biophysica Acta, 444 (1976) 11--22

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27977 FUCOLIPID METABOLISM AS A FUNCTION OF CELL POPULATION DENSITY IN NORMAL AND MURINE SARCOMA VIRUS-TRANSFORMED RAT CELLS

M. GACTO and S. STEINER Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77030 (U.S.A.)

(Received January 12th, 1976)

Summary The incorporation of isotopically labeled fucose into the lipids of normal and murine sarcoma virus-transformed rat cells as a function of cell population density was examined. Wh6n normal cells were seeded at low cell density, the levels of the major fucolipids, i.e., fucolipids III and IV, were substantially reduced, but then they increased as the cells approached confluency. This variation in synthesis of fucolipids III and IV appeared to be primarily related to cell density and not to cell growth. Chase experiments revealed that the reduced level of fucolipids III and IV in sparse normal cells is due to decreased synthesis rather than to increased catabolism. In contrast to the observations with normal rat cells, the high level of fucolipid III and the low level of fucolipid IV in murine sarcoma virus-transformed rat cells was shown to be independent of cell population density.

Introduction One of the cardinal features of cultured normal cells is a reduced growth rate at confluence. In contrast, transformed cells apparently lack the control mechanism to inhibit growth in response to "cell c o n t a c t " or "topo-inhibition". A number of studies have suggested a possible role for the carbohydrate-containing lipids of the cell surface in the regulation of cellular growth [ 1,2]. The status of glycolipids in a wide range of cultured cells transformed by DNA and RNA oncogenic viruses and by chemical carcinogens [3--7] has been reported. Oncogenic transformation by these agents usually results in incomplete elongation of the oligosaccharide moiety of the glycolipid, sometimes with a build-up of a precursor glycolipid. The decreased levels of the more complex glycolipids in response to oncogenic transformation have often been shown to be due to

12 decreased activities of glycosyltransferases [8--10]. The work cited concentrated on the gangliosides and on non-fucose-containing neutral glycolipids. When the neutral glycolipids were examined as a function of cell population density, it was observed that in normal cells the synthesis of certain neutral glycolipids increased at higher cell density [11--14]. The status of the more complex gangliosides as a function of the cell population density is less clear. Densitydependent synthesis of gangliosides in chick cells has been noted [ 5]. However, similar levels of ganglioside glycosyltransferases in normal mouse cells at high and at low density have been indicated [15]. More recently, an increase in a more complex ganglioside of mouse cells at an early stage of cell contact has been described, even though the increase was not as obvious in crowded cell populations [16]. In contrast to the results obtained with normal cells, transformed cells have been shown to lack cell density-dependent synthesis of glycolipids [11--14]. Studies in this laboratory have focused on the incorporation of radioisotopically labeled fucose into the lipid fraction of normal and virus-transformed cell lines. We have previously reported that oncornavirus-transformed cell lines show a marked decrease in the incorporation of radioactive fucose into a chromatographically less mobile fucolipid, i.e., fucolipid IV, often with a concomitant increase in a more mobile fucolipid, i.e., fucolipid III [17]. Subsequent studies employing a temperature-sensitive transformation m u t a n t of murine sarcoma virus revealed that there is a temporal relationship between reduced incorporation of fucose radioactivity into fucolipid IV and the expression of the transformed phenotype [18]. Similar findings of a block in the incorporation of radioactive fucose into fucolipid of comparable low chromatographic mobility were observed in DNA virus-transformed cells [19] and in cultured h u m a n t u m o r cells [20]. In this communication we report the results of studies in which the synthesis of the fucolipids in normal and murine sarcoma virus-transformed rat cells was examined as a function of cell population density. Materials and Methods

Cell culture and harvesting The cell lines used in these experiments include: normal newborn rat kidney cells (NRK), normal rat embryo cells (RE-2), and the derivative virus-producing cell lines (MSV-NRK and MSV-RE-2) transformed by murine sarcoma-murine leukemia virus complex. A clone derived from NRK cells exhibiting a lower saturation density (NRK-2) was also used. The biological properties of this cell line have been described elsewhere [21]. Cells were cultured in glass bottles {surface area approx. 45 cm 2) in Eagle's minimal essential medium supplemented with 10% fetal calf serum and routinely examined for the presence of mycoplasma. Wherg indicated, the above medium was supplemented with either [1-14C]fucose (54 Ci/mol; New England Nuclear) or [6-3H]fucose (13.1 Ci/ mmol; New England Nuclear) at the levels specified. Following the appropriate growth periods, cell monolayers were washed three times with 0.02 M Trisbuffered saline, pH 7.2, and the cells scraped into this buffer. The scraped cell suspensions were centrifuged at 1500 × g for 5 min and the supernatants (de-

13 signated as " c y t o s o l " fraction} were saved. The pellets were washed four times in cold Tris-buffered saline and the lipid extracted.

Lipid extraction and analysis The washed pellets were sequentially extracted at 52°C twice with CHCI3/ CH3OH (2 : 1, v/v) and twice with CHC13/CH3OH (1 : 2, v/v). Subsequent extractions with these solvents failed to yield a significant amount of organicsoluble fucose radioactivity. The lipid extracts were taken to dryness under N2, redissolved in CHC13/CH3OH/pyridine/H20 (15 : 6 : 4 : 1.5, v/v) and chromatographed on silica gel thin-layer plates (Q-5, Quantum Industries, Fairfield, N.J.) in 2-propanol/NHaOH/H~O (7 : 2 : 1, v/v). The plates were scraped in 1-cm bands from origin to solvent front and the radioactivity measured by scintillation spectrometry [17]. A portion of the cytosol fraction from [14C]fucose labeled cells was chromatographed on Q-5 plates in the above system and the fucolipid extract in a parallel lane. None of the components of the cytosol had the same chromatographic mobilities as the major fucolipids, although the mobility of free fucose was similar to that of fucolipid III. In order to exclude the possibility that radioactivity from free fucose was contributing to that of fucolipid III, a portion of the lipid extract was chromatographed on Q-5 plates in CHC13/CH3OH/H20 (60 : 35 : 8, v/v). In this system, fucolipid IlI and free fucose are readily separable, although the cell washing procedure outlined above usually removes all the residual free fucose. In experiments involving long-term [3H]fucose labeling followed by chase, the a m o u n t of labeled precursors, fucose, GDP-fucose and fucose-l-phosphate, was estimated by descending chromatography of the cytosol fraction on Whatman 3M paper (50 cm length) in ethanol/1 M ammonium acetate, pH 7.5 (5 : 2, v/v). To locate the radioactivity, the chromatogram was cut in 1-cm strips from origin to solvent front and the fractions counted. Identification of the radioactive peaks, i.e., fucose, fucose-l-phosphate, GDP-fucose, was based on chromatographic comparison with standards.

Hydrolysis of lipids The radioactivity incorporated into fucolipids III and IV was fucose, as verified by mild acid hydrolysis [ 17] followed by identification of the hydrolysis product by chromatography on analytical cellulose thin-layer plates (Q-2, Quantum Industries}. Two solvent systems, butanol/pyridine/H~O (6 : 4 : 3, v/v) and butanol/propionic acid/H~O (6 : 3 : 4, v/v}, were used. Two minor radioactively labeled components with higher chromatographic mobilities than fucolipids III and IV were also observed, particularly when the tritiated form of the radioactive sugar was used. When subjected to mild acid hydrolysis these minor components did not yield free fucose. These components were detected in the Folch lower fraction of lipid extracts of stock radioactive fuco fucose. They appear to be less than a 0.1% lipophilic contamination of the commercial radio-labeled sugar and have been isolated from [1-3H]fucose, [1,5,6-3H]fucose, and [6-3H]fucose from New England Nuclear and Amersham/Searle Corporations. The "non-fucose" radioactivity represents only a small fraction of the total lipid radioactivity of the cells except under special

14

labeling conditions, e.g., serum deprivation. Upon thin-layer chromatography these contaminants migrate to cm 12 and 17, areas II and I, respectively. Protein determination The a m o u n t of whole cell protein was determined by the m e t h o d of Lowry et al. [22].

Results Previous chromatographic analyses have revealed the presence of two major fucose-labeled lipids in a number of cultured mammalian cell lines. Fig. 1 illustrates the chromatographic pattern of fucolipids from confluent NRK cells and densely growing NRK cells transformed by MSV (MSV-NRK). The relative reduction in incorporation of [3H]fucose into fucolipid IV of MSV-NRK cells with parallel increase in fucolipid [II is clearly evident.

Long-term labeling studies The radioisotopic fucose labeling patterns of normal and transformed rat cell lines at low and high cell population density have now been examined. The initial high cell density [~4C]fucolipid pattern of NRK-2 cells (Fig. 2A) is typical of the pattern obtained with confluent normal cells (Fig. 1). Passage again to high cell density results in essentially the same pattern of incorporation of [~4C]fucose into the two major fucolipids (Fig. 2B). In contrast, passage of a

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F i g , 2. L o n g - t e r m l l d c ] f u c o s e l a b e l i n g p a t t e r n s o f r a t k i d n e y c e i l s as a f u n c t i o n o f cell p o p u l a t i o n d e n s i t y . N R K - 2 c e i l s w e r e u n i f o r m l y l a b e l e d i n m e d i u m c o n t a i n i n g 0 . 5 ktCi o f [ 1 4 C ] f u c o s e / m l o f m e d i u m , h a x v e s t e d a t c o n f l u e n c y a n d p a s s a g e d a t h i g h ( 1 - - 2 * 1 0 6 c e l l s / c u l t u r e ) o r l o w ( 2 - - 3 • 1 0 5 c e l l s / c u l t u r e ) cell population density. The alternative radioisotopic form of fucose, i.e., 3H, yielded essentially the same q u a l i t a t i v e r e s u l t s . W h e n p a s s a g e d f r o m l o w t o h i g h cell d e n s i t y o r t h e r e v e r s e , t h e c e i l s w e r e r e m o v e d w i t h t r y p s i n , p o o l e d a n d s e e d e d a t t h e a p p r o p r i a t e d e n s i t y a n d h a r v e s t e d 4 8 h a f t e r s e e d i n g . Subcont~luence was achieved by continued growth of low density cells. The fucolipid patterns of confluent versus s p a r s e c e l l s w e r e e x a m i n e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s .

Fig. 3. L o n g - t e r m [ 1 4 C ] f u c o s e labeling p a t t e r n s o f R E - 2 cells as a f u n c t i o n o f cell p o p u l a t i o n d e n s i t y . Equivalent numbers of RE-2 cells were used to seed sparse and dense cultures to those indicated in Fig. 2 for NRK-2 cells, and the cells were also harvested 48 h after passage. For details of fucolipid extraction and analysis, see Materials and Methods.

16 portion of the high density grown cells to low cell density results in a significant decrease in the total fucose radioactivity (Fig. 2C), and the decrease appears to be primarily due to a reduction in the a m o u n t of fucolipid IV. Sequential passage of low density grown NRK-2 cells again to low density (Fig. 2E) results in an even greater reduction in total fucolipid radioactivity, and this diminution correlates with a decrease in the radioactivity in both fucolipids III and IV. Conversely, when sparsely growing NRK-2 cells are seeded at high density (Fig. 2D), the levels of fucolipids III and IV are comparable to those of densely growing cells. Sparsely seeded cells grown to subconfluence (Fig. 2F) show noticeably less radioactivity in fucolipid IV as compared to confluent cells. However, if the subconfluent cultures were allowed to grow to confluence, the fucolipid patterns were observed to be comparable to the confluent cells (data not shown). Trypsinization of cells had no effect on the fucolipid composition as judged by a comparison of trypsinized and scraped cells. Like studies with RE-2 cells seen in Fig. 3 yielded similar results to those ohserved with the NRK-2 cells. It can be concluded from the above observations that sequential passage of normal rat cells at low density results in a reduction in the a m o u n t of complex fucolipids (i.e., III and IV). The status of the fucolipids in dense versus sparse cell cultures of MSVtransformed rat cells, after long-term labeling, are summarized in Fig. 4. The fucolipid patterns of sparse versus dense cultures of the transformed cells are essentially the same, indicating a lack of cell density-dependent synthesis.

Influence of the stage of growth on fucolipids Because normal confluent cells manifest a marked decrease in growth rate as compared to sparse cells, it was important to clarify whether the differences in the levels of fucolipids III and IV were due to " g r o w t h " versus " n o n - g r o w t h " or to density per se. A growth curve of NRK cells at low density in reduced serum as compared with growth in medium containing 10% fetal calf serum is seen in Fig. 5. Approximately 15 h after changing to low serum medium the sparsely seeded cells were observed to be stationary, while those in 10% fetal calf serum continued exponential growth. Maintenance of the sparse NRK cells for a longer time (i.e., 39 h) under serum-deprived conditions did not result in significant cell perturbation as judged by the fact that following addition of 10% fetal calf serum supplemented medium there was a rapid initiation of growth comparable to cells continuously maintained in 10% fetal calf serum supplemented medium. Examination of the fucolipid pattern o f stationary sparse cells (Fig. 5F) reveals a dramatic decrease in the level of fucolipids III and IV as compared to stationary confluent cells (Fig. 5A). Likewise, the rapidly growing sparse cells (Fig. 5D, E) show a decrease in these components. From these data we conclude that both growing and stationary low population density normal cells have significantly reduced levels of fucolipids III and IV. The possibility~that a serum deprivation effect unrelated to quiescence might be responsible for the inhibited synthesis of fucolipids III and IV in stationary cells, at low population density, was also examined. NRK cells seeded at high density were serum-deprived and the fucolipid pattern analyzed. The results (Fig. 5C) show that the levels of fucolipids III and IV in serum-deprived high cell density cultures is comparable to non-serum-deprived high cell density

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FRACTIONNUMBER F i g . 4. L o n g - t e r m [ 3 H ] f u c o s e l a b e l i n g p a t t e r n s o f M S V - N R K c e l l s a s a f u n c t i o n o f t h e ceil p o p u l a t i o n density. MSV-NRK cells were labeled to uniform specific activity in the presence of 2 pCi of [3H]fucose/ ml of culture medium. Dense cultures were seeded at 1.5 • 107 cells/culture bottle and sparse cultures were seeded at 4 • 105 ceils/culture bottle; the cells were harvested 48 h after seeding. In addition, the medium was changed twice daily to minimize the effect of rapid glucose utilization. For details of scheme o f p a s s a g e t o h i g h o r t o l o w cell p o p u l a t i o n d e n s i t y , s e e F i g . 2. E x p e r i m e n t a l d e t a i l s f o r f u c o l i p i d e x t r a c tion and analysis are described in Materials and Methods.

(Fig. 5B) or t o c o n f l u e n t (Fig. 5 A ) cultures. H e n c e , serum deprivation by itself d o e s n o t appear t o result in t h e inhibited s y n t h e s i s o f f u c o l i p i d s III and IV. High and l o w cell d e n s i t y M S V - N R K cells w e r e also labeled for 16 h. T h e a m o u n t and d i s t r i b u t i o n o f t h e f u c o l i p i d s w e r e c o m p a r a b l e at b o t h densities

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F i g . 5. [ 3 H ] f u c o s e l a b e l i n g p a t t e r n s o f n o r m a l cells a t v a r i o u s s t a g e s o f g r o w t h . N R K cells w e r e s e e d e d in c o m p l e t e m e d i u m a t h i g h o r a t l o w cell p o p u l a t i o n d e n s i t y t o o b t a i n t w o s e r i e s o f c u l t u r e s . C e l l s w e r e allowed to attach overnight and then the medium from a number of cultures in each series was removed, the cells washed with warm Tris-buffered saline and the incubation continued in medium containing 0.5% fetal calf serum. The remainder of the cultures were maintained in complete medium with 10% fetal calf serum. The media were supplemented with 5 pCi of [3H]fucose/ml. Each fucolipid profile repr e s e n t s 1 6 h o f l a b e l i n g . ( X ) s h o w s t h e s t a g e s o f g r o w t h a t w h i c h cell c u l t u r e s w e r e s a m p l e d . T h e s t r a i g h t arrow indicates the change to serum-deprived medium; the wavy arrow indicates the time at which serumstarved cultures were again incubated in medium with 10% fetal calf serum. (Y) shows the [3HI labeled f u c o l i p i d p a t t e r n s o b t a i n e d f r o m N R K cells a t d i f f e r e n t s t a g e s o f g r o w t h , i . e . , ( A ) , c o n f l u e n t q u i e s c e n t ; (B), high density growing; (C), high density serum-deprived stationary; (D), subconfluent; (E), low density growing; (F), serum-deprived stationary sparse cells. It should be noted that the NRK cells grow to higher s a t u r a t i o n d e n s i t y t h a n t h e N R K c l o n e 2 c e l l s t h a t w e r e u s e d in t h e e x p e r i m e n t s h o w n i n F i g . 2. T h e N R K cells were utilized in this study because of their hazdiness under conditions of serum deprivation. Details for fucolipid extraction and analysis and origin of components a t c m 1 2 a n d 17 are g i v e n i n M a t e r i a l s

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10 15 0 5 FRACTION NUMBER F i g . 6. [ 3 H ] f u c o s e l a b e l i n g p a t t e r n s o f M S V - N R K c e l l s a t h i g h v e r s u s l o w cell p o p u l a t i o n d e n s i t y . M S V N R K c e l l s w e r e l a b e l e d a t h i g h ( 4 . 0 - 1 0 7 c e l l s ] c u l t u r e ) o r l o w ( 3 . 7 • 1 0 5 c e l l s ] c u l t u r e ) cell p o p u l a t i o n d e n s i t y i n m e d i u m s u p p l e m e n t e d w i t h 5 / ~ C i o f [ 3 H ] f u c o s e l m l o f m e d i u m f o r 1 6 h a s i n d i c a t e d i n F i g . 5. T h e f u c o l i p i d p a t t e r n s o f c r o w d e d a n d s p a r s e c u l t u r e s w e r e o b t a i n e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s .

{Fig. 6). These results, like those with uniformly fucose-labeled MSV-NRK cells (Fig. 4), indicate that the metabolism of the fucolipids of transformed cells is independent of the cell population density. Chase studies The decreased levels of fucolipids III and IV in sparse normal cells suggest either a block in synthesis or an increase in catabolism. To differentiate between the two possibilities, normal cells were labeled for long term with [3H]fucose and then chased at high and low cell density. Within 6 h after the initiation of the " c h a s e " period, the radioactivity in fucose, GDP-fucose, and fucose1-phosphate was too low to be detected in the cytosol fraction of the cells, indicating an effective chase of the precursor pools. High and low cell density cultures were harvested 40 h after initiation of the chase {Fig. 7). The results show that there was no demonstrable loss of fucolipid IV following the chase at low cell density. The above results are consistent with the interpretation that there is not enhanced catabolism of fucolipids at low cell density and that the decreased incorporation of radio-labeled fucose into fucolipids III and IV is probably due to a decrease in synthesis. Comparable findings were obtained using NRK cells. A chase experiment with MSV-NRK cells revealed that the

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FRACTION NUMBER Fig. 7. C h a s e o f l o n g - t e r m [ 3 H ] f u c o s e l a b e l e d R E - 2 cells at h i g h a n d at l o w cell p o p u l a t i o n d e n s i t y . R E - 2 cell c u l t u r e s w e r e l a b e l e d f o r 72 h in m e d i u m s u p p l e m e n t e d w i t h [ 3 H ] f u c o s e (5 # C i / m l ) a n d c o l l e c t e d at c o n f l u e n c y . T h e f u c o l i p i d p a t t e r n o f o n e - t h i r d of t h e s e cells f o l l o w i n g t r y p s i n i z a t i o n w a s d e t e r m i n e d . A s e c o n d p o r t i o n o f cells w a s s e e d e d in " c h a s e " m e d i u m at h i g h cell d e n s i t y ( a p p r o x . 1.5 • 106 c e l l s / c u l t u r e ) a n d t h e final t h i r d w a s s e e d e d in " c h a s e " m e d i u m at l o w cell d e n s i t y ( a p p r o x . 2 • 105 c e l l s / c u l t u r e ) . T h e c h a s e m e d i u m was, E a g l e ' s m i n i m a l e s s e n t i a l m e d i u m s u p p l e m e n t e d w i t h 3 . 8 . 1 0 - 7 M L - [ 1 2 C ] f u c o s e . B is t h e f u c o l i p i d p r o f i l e o b t a i n e d f r o m d e n s e cells f o l l o w i n g 4 0 h of c h a s e . C is t h e f u c o l i p i d p r o f i l e obt a i n e d f r o m s p a r s e cells f o l l o w i n g 4 0 h o f c h a s e . F o r e x t r a c t i o n a n d a n a l y s i s see M a t e r i a l s a n d M e t h o d s . F i g . 8. Chase o f l o n g - t e r m [ 3 H ] f u c o s e l a b e l e d M S V - N R K cells. M S V - N R K cells w e r e l a b e l e d f o r 72 h, h a r v e s t e d a n d s e e d e d at 5 • 106 c e l l s / c u l t u r e in c h a s e m e d i u m . T h e a b o v e p r o f i l e s r e p r e s e n t t h e f u c o l i p i d p a t t e r n s o f M S V - N R K cells l a b e l e d f o r 72 h ( t o p ) a n d a f t e r 4 0 h of c h a s e ( b o t t o m ) . F o r e x p e r i m e n t a l details see F i g . 7.

21 major fucolipids of transformed cells, like those of normal cells, do not turn over rapidly (Fig. 8). Discussion The results of this study are consistent with the interpretation that the synthesis of fucolipids III and IV is cell population density-dependent in normal rat cells. At low density, whether the cells are growing or quiescent, the synthesis of these components is dramatically decreased as compared with high density cells. These findings would favor the concept that the effects are due to cell density per se [13]. Although direct evidence cannot be cited to show that the synthesis of fucolipids III and IV in normal cells is in response to cellto-cell contact, the data are consistent with this interpretation [23]. In fact, when contact inhibited dense cells are passaged to low cell density, the decreased ability to synthesize fucolipids III and IV occurs rapidly. In contrast, shift from low to high cell density results in initiation of synthesis. The chase results indicate that the catabolism of fucolipids III and IV is not rapid. Once synthesized at high cell density, these components are apparently diluted out as a function of cell division following shift to low cell density. Recent studies in which neutral glycolipids of hamster cells were examined following shift from high to low density also support the idea that complex glycolipids synthesized at high cell density are not rapidly lost following shift to low cell density [13,24]. Hence, the synthesis per se of the more complex fucolipids and other glycolipids, in response to increased cell density, may be linked to growth regulation. The cell density effects on fucolipid synthesis in normal cells may reflect altered levels of synthetic enzymes [8--10], density-dependent regulation of the activity of these enzymes, or changes in the nucleotide-sugar precursor pools [25]. Further study is needed to pinpoint the molecular basis for densitydependent synthesis of fucolipids III and IV. Whichever explanation is the correct one, the regulation of fucolipid synthesis in MSV-transformed cells appears to be quite different. Not only are there quantitative differences in the levels of the fucolipids [17--20], but also their synthesis is apparently not regulated as a function of cell density. One important implication of the chase studies with the MSV-NRK cells is that the decreased level of fucolipid IV in transformed cells is due to inhibited synthesis rather than increased catabolism. We have also found that murine leukemia virus-infected but not transformed NRK cells display changes in the fucolipid pattern in response to cell density similar to those observed with NRK cells (unpublished data). Thus, the lack of cell density-dependent synthesis of fucolipids III and IV is apparently related to virus transformation and n o t solely to virus infection. There is at least one feature of the cell density regulation of fucolipids that differs somewhat from other glycolipids. In general, it has been observed that the glycolipids whose synthesis is cell density-dependent are the ones that are decreased following transformation [26]. We have observed that the synthesis of both fucolipids III and IV is reduced at low cell density. Thus, the primary metabolic block in fucolipid metabolism of normal cells in response to low cell density appears to differ from that observed when these cells are transformed by murine sarcoma virus.

22 Fucolipids with similar chromatographic properties to those described in this study have been observed in all the cultured mammalian cell lines examined {human, mouse, rat, baboon and hamster) [17--20]. We are in the process of determining if cell density-dependent synthesis of fucolipids III and IV is a ubiquitous feature of normal mammalian cells and if the loss of this densitydependent synthesis is characteristic of both DNA and RNA virus-transformed cells. In addition, recent studies [27] suggest that fucolipids III and IV are unusual in that they contain a sphingosine-like base that lacks an amide-linked fatty acid typical of most other glycosphingolipids. In order to gain insight into the metabolic steps involved in their synthesis, we are currently analyzing the structures of fucolipids III and IV.

Acknowledgments The authors would like to acknowledge the excellent technical assistance of Ms. Y.S. LaBelle. We would also like to acknowledge the helpful suggestions of Dr M.R. Steiner and Dr J.L. Melnick during the course of these studies. The work was supported in part by research grants CA 16,311 and CA 10,893 from the National Cancer Institute, National Institutes of Health. M. Gacto is a Fellow of the Spanish Cancer Society. S. Steiner is the recipient of Faculty Research Award FRA-131 from the American Cancer Society, Inc.

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Fucolipid metabolism as a function of cell population density in normal and murine sarcoma virus-transformed rat cells.

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