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Experimental Cell Research 106 (1977) 79-87

PHOSPHOLIPID

DEGRADATION MURINE

BY SV40-TRANSFORMED

FIBROBL’ASTS

D. FAN and H. VOELZ Department of Microbiology, School of Medicine, Medical Center, West Virginia University, Morgantown, WV 26506, USA

SUMMARY Simian virus-transformed mouse fibroblasts (SV3T3), but not the parent 3T3 cell line, can grow on and degrade phospholipid films made from sheep brain extracts. The SV3T3 cells degrade phospholipids dispersed in the medium only when previously grown on phospholipid film. Both cell lines grow in conditioned medium made from SV3T3 cultures thdt were grown on a phospholipid film. During growth of SV3T3 on phospholipid films, or during growth of either cell type in conditioned medium, neutral lipids accumulate as droplets in the cytoplasm. Multinucleate cells are then formed at approaching confluence.

ivanova & Margolis ([l] and personal communcation) developed a system which appeared to be suited for the investigation of the interaction between cultured cells and artificial membranes. A phospholipid extract from calf brain was made into a thin film onto which cells were planted, and the interaction between both film and cells could be observed. The cells, including a SV40-transformed epithelial kidney cell line, grew only on that portion of cover glasses not coated with the phospholipids. The cells did not degrade the film, and their lifespan did not exceed 3 days. When we tested our 3T3 and SV3T3 cell lines on phospholipid films, we obtained different results. We found that the latter but not the former cell line degraded the phospholipids. In this report we describe the growth pattern of the cell lines on phospholipid films and present evidence for its degradation by the SV3T3 cells. 6-771816

MATERIALS

AND METHODS

Cell lines and growth conditions The Swiss 3T3 cell line used in these studies was purchased from Flow Laboratories, Rockville, Md. A SV3T3 line was derived from this line by transformation with SV40 virus, strain A2895 provided by Dr B. E. Kirk of this Department. Todaro $r Green’s standard procedure for viral transformation was followed, and their criteria for a transformed cell line served to characterize the transformant. Both cell lines were maintained routinelv. free of mycoplasma, in 3012 tissue culture flasks (Falcon, Oxnard, Calif.), in Dulbecco’s modified Eagle’s medium (GlBCo, Grand Island, N.Y.), plus 5Q @g/ml gentamicin (Schering, Port Reading, NJ.) and 10% calf serum (GlBCo). The cells were cultured at 37°C in a 5 % CO, incubator.

Phospholipidfilms Phospholipid films were formed on Petri dishes or cover glasses using the method of Ivanova & Margolis [l]. Principly, the lipids were extracted from the white matter of sheep brains with chloroform-methanoi according to Folch et al. 121.The dried extracts were redissolved in benzene (25 mglml) and aseptically placed on the glass surface by evaporation in a continuous vacuum.

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Fan and Voelz

Autoradiography

Lipid analyses

For autoradiography, cells were grown for 4 days in 0.3 uCi/ml l*‘UTdR (snec. act. 59.5 mCi/mM: Schwartz/M&n, Grange&g, N.Y.). The labeled ceils were then combined with an equal number of nonlabeled cells of the same age. This mixed population was grown on cover glasses coated with phospholipid films until multinucleate cells were formed. The cells on cover alasses were washed in ahosnhate buffer saline and processed for autoradiography: The cells were fixed in ethanol/acetic acid (3 : 1). The air-dried cover glasses were dipped in llford L4 emulsion (Ilford, Essex, UK) diluted to obtain a monolayer film. After exposure from 3 to 14 days, the preparations were developed in D-19 (Kodak, Rochester, N.Y.) and fixed. Multinucleate cells were observed and counted under a phase contrast microscope (Carl Zeiss, Oberkochen, BRD).

Cells with accumulated lipids were homogenized and the cytosol fraction (100000 g) was added to a silicic acid column. The neutral lipids were eluted with ethyl ether/hexane 1: 1 (v/v) and analysed by the procedures of Mackenzie et al. [6]. For the determination of lipids and phospholipids, cell samples were removed from culture flasks or Petri dishes-with a rubber policeman, washed and suspended in cold phosphate-buffered saline, and homogenized at 4°C either with a handoperated ground-glass Pyrex tissue grinder or a Teflon Potter-Elvehjem homogenizer. The unbroken cells were removed bv centrifugation at 500 .afor 5 min. In some experiments, the homogenates were separated into soluble fractions and cell nellets bv centrifugation at 10000 g in a Beckman SW50.1 rotor using aBeckman L5-65 ultracentrifuge (Beckman, Belmont, Calif.). The lipids were extracted from the homogenates with chloroform-methanol [Z]. Homogenate samples were analysed for total lipid, or were separated into neutral lipids and phospholipids with a silica gel G column. The neutral lipid and phospholipid fractions were eluted with 25 ml chloroform and 45 ml methanol, respectively. The solvents were evaporated at 45°C by a Buchi Rotavapor under nitrogen (Ruico Instrument, Greenville, Ill.). Lipids were extracted from the media by the same procedures. The individual lipids were analysed by established procedures [7]. Chromdtographed lipids were located by iodine vapor or dichlorofluorescein spray, phospholipids were detected by molybdate phosphate reagent; those with free amino groups were identified by ninhydrin spray, and those containing choline by Dragendorfs reagent. Other phospholipids and neutral lipids were identified by comparison with known standards. The lipids were analysed by the following TLC systems.

Radioactive

labeling

The DNA/protein ratio was determined by incubation of arowine cells in the oresence of 0.3 /-0/ml l14C1TdR (Sphwarg/Mann, Orangeburg, N.Y.). dells-growing on a phospholipid film deposited on cover glasses were compared with those growing in normal medium. Before harvesting, the multinucleate cells were counted under a Zeiss phase contrast microscope. At 24 h intervals the washed cells were scraped from the cover glasses with a rubber policeman and homogenized with a ground-glass, hand-operated tissue grinder (Bellco, Vineland, N.J.). Protein determinations were by the method of Lowry et al. [3]. Determination of radioactivity was made by placing the homogenate samples (0.2-0.4 ml) in 10 ml of a scintillation cocktail (2 liters toluette; -1 1 Triton X-100; 5 g 1,4Bis-(Z-(S-phenyloxazolyl))-benzene; 16.5g 2$diphenyloxazole; 333 ml water). The samples were counted for 10 min each in a Packard Tri-Carb scintillation spectrometer, model 3320 (Packard, Downers Grove, Ill.).

Electron microscopy For electron microscopy, cells were harvested by trypsinization,pe&eted and-processed aqsonling to Saba&i et al. [4] and Palade [5’], using buffered 2.5% glutaraldehyde (Kodak, Rochester, N.Y.) and 1.O% osmium tetroxide (Biodynamics, Rockville, Md). Specimens were embedded in Epon and thin sections were stained with 2.0% aqueous uranyl acetate and lead citrate. The sections were examined with an RCA EMU3G electron microscope.

Inhibitor

of mitosis and cytokinesis

SV3T3 cells were grown on phospholipid films until morphological changes were observed (lipid accumulation and multinucleation). They were then returned to normal growth conditions. After the cells had reverted to normal morphology, they were divided into two groups and transferred onto phospholipidcoated Petri dishes. One group obtained either 5.0 Fg cytochalasin B (CB) (Aldrich Chemical, Milwaukee, Wise.), or 1O-6M colchicine (Eli Lilly, Indianapolis, Ind.) per ml of medium. The second group was the untreated control. The cells were observed every 3 h for morphological changes. Exp Cell Res IO6 (1977)

Lipids were separated on 0.5 mm Silica gel GF-254 (EM, Elmsford, N.Y.) plates. The plates were developed by a one-dimensional system with chloroform/methanol/water (65 : 25 : 4 v/v).

To&d lipids.

Lipids were separated on 0.5 mm Silica-gel GF-254 plates by a two-dimensional system, developed in the first direction with chloroform/ methanol/ammonia (65 : 40 : 5 v/v) and in the second direction with chloroform/methanol/acetone/acetic acid/water (33 : 5 : 36 : 10: 1 v/v).

Total phospholipids.

Neutral

lipids

of soluble fractions

and cell pellets.

These lipids were determined against a neutral lipid standard (Hormel no. 2) at 25 mg/ml. The lipids were separated on 0.25 mm silica gel G plates. The plates were developed by a one-dimensional system with petroleum ether/ether anhydrous/acetic acid (100 : 10 : 2 v/v). lipids of conditioned media. The lipids were separated on 0.25 mm silica gel G plates and developed as described in the previous section.

Neutral

Phospholipids of conditioned media and lipid extracts from sheep brain. The lipids were separated on 0.25

mm silica gel H/magnesium silicate plates. The plates were developed by a two-dimensional system as de scribed in the section on total phospholipids.

Phospholipid degradation

Fig. 1. Edge of spreading colony (urrows) of SV3T3 cells on a phospholipid film. Lipid droplets are seen as refractile cytoplasmic inclusions. All light optical observations were made on unstained preparations. x781.

RESULTS Cell growth and multinucleation Two weeks after transfer of SV3T3 cells onto a phospholipid film, the cells appeared to be growing on the film, they began to multiply, and they accumulated refractile cytoplasmic inclusions (fig. 1). The plating efficiency after the initial transfer of SV3T3 cells was estimated to be better than 90%. The possibility of selecting a phospholipid degrading subculture may be ruled out, therefore. The areas around the cells became less opaque. The inclusions stained differentially with Oil Red 0 [8] and we determined the contents as neutral lipids by the procedures of Mackenzie et al. [6]. In electron micrographs, the inclusions were typical for lipid droplets in mammalian cells (fig. 2). When subcultured on phospholipid films, the cells immediately began to grow and degrade the film. At subconfluent growth, up to 60% of the population became multinucleate (fig. 3). The size of the multinucleate cells and the size of the nuclei. was

larger than normal. All multinucleate as well as mononucleate cells contained numerous lipid droplets. In addition, uneven numbers of nuclei were often present in these cells. When the phospholipid film was degraded and the spent medium was replaced with normal medium for growth, the multinucleate cells rounded up as if undergoing mitosis and within 18-24 h only mononucleate cells were seen. During further growth, the cytoplasmic inclusions gradually dissipated. After one more week, the cells were morphologically indistinguishable from non-phospholipid-grown SV3T3 cells, but these cells retained their ability to grow on and degrade a phosphohpid film without the two week lag period we observed when the cells were first planted. A transfer of phospholipid-grown cells onto a fresh phospholipid film had no effect on their growth pattern. In contrast to SV3T3 cells, a 3T3 culture would not grow under any conditions on phospholipid films. However, 3T3 cells, as well as SV3T3 cells, grew in the presence of conditioned medium, i.e. medium from a SV3T3 culture previously grown on a phospholipid film (fig. 4). Under such conditions all the 3T3 and SV3T3 cells accumulated lipids and about 60 o/Gof the population became multinucleate. They reverted to their normal growth pattern after transfer to fresh medium. The 3T3 and SV3T3 cells not grown on phospholipid films were unable to degrade phospholipids added as an aqueous dispersion to the medium. Neither were hpid droplets formed nor were rn~~t~~~~~~ate cells seen. Only SV3T3 cells previously grown on a phospholipid film were able to degrade dispersed phospholipids, accumulate droplets, and become multinucleate. An experiment was done to determine Exp Ce!/ H~.s IO6 (1977)

82

Fan and Voelz

2. Electron micrograph of phospholipid-grown SV3T3 cell? showing I_argeand small lipid droplets (L) in the cytoplasm. X 12239.

Fig.

whether cell fusion or multiple nuclear divisions without cytokinesis produced the multinucleate cells. SV3T3 cells were pulsed with [14C]TdR and mixed with an equal number of non-labeled cells of the same age. The mixed population was grown on a phospholipid film and processed for autoradiography. The 14C label produced densely labeled nuclei in our autoradiograms which facilitated counting of a large number of cells. In the autoradiograms of combined populations grown to the multinucleate stage we did not find any cells with heterogeneously labeled nuclei (fig. 5). In addition, we determined the DNA/proExp Cell Res 106 (1977)

tein ratio of lipid-grown versus normally grown cells. This ratio was found to increase 3-fold in the lipid-grown population (PLSV) during the process towards multinucleation, whereas in mononucleate cells (SV) the ratio remained close to one. This increase of DNA/protein ratio was concomitant with the increase in the number of multinucleate cells (fig. 6). To test further whether multiple nuclear divisions and not cell fusion was the cause of multinucleation, experiments were performed with the mitotic inhibitor colchicine, and the cytokinesis inhibitor cytochalasin B (CB), in cells which were planted on a phos-

Phospholipid

degradation

8.7

Fig. 3. Multinucleate SV3T3 cells at approaching confluence of growth on phospholipids. Cells contain numerous lipid droplets in their cytoplasm. Note the increase in cell and nucleus size. x781. Fig. 4. Sub-confluent 3T3 cells grown for 24 h in conditioned medium from a phospholipid-grown SV3T3 cuiture. X781. Fig. 5. Autoradiogram of a mixed population of nonlabeled and PClTdR labeled SV3T3 cells. Cells were grown on ihos-pholipid-coated cover glasses for 3 days. Long arrow: homogeneous, labeled nuclei in multinucleate cells; short arrow: homogeneous, nonlabeled nuclei. ~781.

pholipid film. In agreement with the results with autoradiography, no multinucleate cells formed on lipid films when colchicine was added to the medium (not shown). The effect of CB is shown in fig. 7. All cells became multinucleate. Although most of these cells contained only two nuclei, some trinucleate cells were also seen. Lipid degradation

The most siginifkant changes in total lipids in cells grown on a phospholipid film are shown in fig. 8. We found two unidentified lipids in these cells (B; a and b) which were not present before growth on phospholipids (C). One of the lipids (h) was found in the

phospholipid film (A), i.e. sheep brain extract. There were also qualitative differences in the lipids of the culture fluid from cells grown on phospholipids. These changes can be seen in the chromatogram of neutral lipids in fig. 9. Cholesterol esters (c) and triglycerides (a) were present in the conditioned media of cells after growth on the film but both were absent in the spent medium (A) from cells grown in Dulbecco’s modified Eagle’s medium. The neutral lipids present in cells before and after growth on phospholipids and their distribution in the 10000 g pellet fraction and the supernatant solution are summarized in fig.

84

Fan and Voelz

Fig. 6. Abscissa: time (days); ordinate: (left) multinucleate cells (X 1000); (right) radioactivity (IO5 cpm/ mg protein). [W]TdR uptake by SV3T3 cells growing in normal medium (SV, O-O), and SV3T3 cells growing on phospholipid film (PLSV, 0-O). MNSV (A-A), multinucleate cells/cover ghassof phospholipid-grown culture.

10. No cholesterol was found in SV3T3 cells grown in normal medium or on phospholipids (B; PL-B). Cholesterol was found in 3T3 cells grown in normal medium (A) but was absent in the supernatant fraction of 3T3 cells grown in the conditioned medium of phospholipid-grown SV3 T3 cells (PL-A; a).

A change in the distribution of cholesterol esters among the fractions of both cell lines also occurred. During growth of SV3T3 cells on the phospholipid films, cholesterol esters were absent in the pellet fraction (PL-I?; b). In 3T3 cells grown in phospholipid-conditioned media, the cholesterol esters were present in the pellet fraction (PL-A; b) but absent in the supernatant fraction (PL-A; a). In addition to these changes, triglycerides were found only in the supernatant fractions of 3T3 cells grown in normal medium (A; a). Free fatty acids were detected exclusively in the pellet fractions of conditioned medium grown 3T3 cells (PL-A; b) and phospholipid grown SV3T3 cells (PL-B; b). The major components of the phospholipid film (sheep brain extracts) are shown in fig. 1I A. They are phosphatidyl ethanolamine, phosphatidyl choline, and phosphatidyl serine. The phospholipids con-

B 0 8 e;

4 00 Fig. 7. Growth of SV3T3 cells on phospholipid film in the presence of CR. Arrows, cells with more than two nuclei/cell. Unstained. x625.

Fiig. 8. Onedimensional TIC of total lipids. (A) Sheep brain lipid extracts; (B) phospholipid-grown SV3T3 homogenate; (C) SV3T3 homogenate from cells not grown on phospholipids. 0, origin; PC, phosphatidyl choline; PI?, phosphatidyl cthanolamine; a, h, extra lipids.

v

t3

Phospholipid

-

-

P

I

degradation b

85

-1

:

0 8

0

6 0 8

0 4

0

3

0 0

Fig. 9. One-dimensional TLC of neutral lipids of medium from: (A) SV3T3 culture; (B) phospholipidgrown SV3T3 culture. c, Cholesterol esters; a, triglycerides.

tained in the normal growth medium, which were added with the serum supplement, were essentially the same major components as those of the phospholipid film (fig. 11B). Lysophosphatidyl choline (LPC) was present in the phospholipid film and in normal medium. After growth of SV3T3 cells on the film in normal medium, most of the phosphatides of the medium had disappeared (fig. 11C). Lysophosphatidyl choline was no longer detectable and phosphatidyl choline, in reduced quantities (not shown), remained. Most of the unidentified lipids of the film as well as those present in normal medium disappeared. Instead, phosphatidic acid was found in the conditioned

0

0

2I 80 -8 -

0 f -

0

0

0

A

?L.A +-

l3

I 1P

0 PL-: -

Fig. 10. One-dimensional TLC of neutral lipids. S, Hormel neutral lipid standard; 1, diglycerides; 2, cholesterol; 3, fatty acids; 4, triglycerides; 5, cholesterol esters. A, 3T3 homogenate; PL-A, medium kduced 3T3 homogenate; B, SV3T3 homogenate; FL,-B, phospholipid-grown SV3T3 homogenate; a, supernatant solution; b, pellet; c, triglycerides; d, fatty acids.

medium. There was no change in the major phospholipids of the cells after growth on phospholipids (not shown). DISCUSSION

To our knowledge, degradation of a phospholipid film by cultured cells has not been reported before. There is little doubt that our SV3T3 line can grow on and degrade such a film. Besides direct observation of growing cells, this is evidenced by (I) the Fig. II. Two-dimensional TLC of phospholipids of conditioned media and of lipid extracts from sheep brain. (A) Sheep brain; (B) SV3T3 control; (C) phospholipid-grown SV3T3; a, phosphatidic acid;LPC, lysophosphatidyl choline; PC, phosphatidyl choline;PE, phos-

86

Fan and Voek

acquisition by the cells of two extra lipids during growth on the tilm; (ii) the appearance of cholesterol esters, triglycerides, and phosphatidic acid in the growth medium; and (iii) disappearance of most of the phosphatides from the phospholipid substrate, i.e. degraded phospholipid film and the lipids added with the serum supplement. One of the acquired lipids was a component of the phospholipid film and may have been taken up in an unmodified form by the cells. The second lipid was either (i) a breakdown product of film degradation and was taken up by the cells unmodified; or (ii) it was synthesized by the cells during growth on lipids. The serum supplement was probably not the source because no extra lipids were found in cells when grown in normal medium. Although the chemical nature of the extra lipids was not determined, they were neutral lipids, and at least one of them was a component of the cytoplasmic inclusions. They were not triglycerides, the usual storage material in a large variety of other cell types. This is important because triglycerides are also found in sublines of the original 3T3 line [9] and are major storage lipids when fibroblasts convert to adipocytes [lo]. Besides phosphatidic acid and cholesterol esters, triglycerides were found only in conditioned media when the cells grew on phospholipids and in the supernatant fraction of 3T3 cells grown in normal medium. Phosphatidic acid was probably a degradation product of one of the phosphatides. It was found in surprisingly small quantities considering the relatively large amounts of major phosphatides which disappeared from the substrate. It can be excluded as a storage lipid because it was not found in cells under any growth conditions. The same may apply to cholesterol esters which were absent in supernatant cell fractions of 3T3 cells Err, Crll Res IO6 (1977)

grown in media from phospholipid-grown SV3T3 cells. Under these growth conditions, 3T3 cells usually accumulate lipids of the same type as the transformed line, i.e. neutral lipids. Cholesterol was absent in our SV3T3 cells, regardless of growth conditions and substrate. Possibly this cell line lacks cholesterol esterases. This would account for why only esters are found in these cells, and also why the cholesterol esters in the conditioned medium cannot be de-esterified . Cells with multiple nuclei have been frequently observed in tumors [ 111,normal tissues [12], and in cultured cells infected with various viruses [ 13, 141. Multinucleation can be also experimentally induced by viruses [ 151, lipids [ 161, lysophosphatides [17], and with CB [18]. Multinucleate cells are known to be produced by cell fusion [I.51 and by failure of cytokinesis after normal [9] or simultaneously multiple mitosis [ 191.Technical difticul ties, such as high cell densities at areas where multinucleation was in progress, or swift migration prevented observation of multiple mitotic figures or cell fusion in our cultures. However, the rate at which cells became multinucleate was time dependent, and the number of multinucleate cells was limited within a population to no more than 60%, facts which argue against cell fusion. Also arguing against fusion are the data from the autoradiography experiments, the increase in the DNA : protein ratio during multinucleation, and the failure to detect multinucleate cells under conditions of mitotic arrest whereas multinucleation proceeded in the presence of a cytokinesis inhibitor. In other studies, only binucleate cells were formed in a 3T3 subline which accumulated triglycerides in the cytoplasm [9]. The lipids apparently delay cyto-

Phospholipid

kinesis, because multinucleation did not proceed beyond two nuclei/cell as the lipids became diluted in the progeny. In our system, degradation of phospholipids continued during cell growth and more-not less-breakdown products for uptake and storage became available. An increase in storage lipids may have caused the number of nuclei/cell to increase rather than to decrease at approaching confluence. Also, the cells can be held at that stage as long as lipids are not diluted by fresh medium. Moreover, ceils not degrading phospholipids (3T3) store lipids and become multinucleate when growing in conditioned media from phospholipid-grown SV3T3 cells. These results suggest that the cells are triggered into multinucleation during accumulation of neutral lipids but not during phospholipid degradation. This work was supported by a grant from the WVU Medical Corporation. We thank Dr Brooks for technical advice, and Drs Charon and Snvder for reading the manuscript.

REFERENCES

degradation

87

3. Lowry, 0 II, Rosebrough, N J, Fan, A L & Randall, R J, J biol them 193(1951) 265. 4. Sabatini, D D, Bensch, K G & Barnett. R .I, J histochem cytochem 10 (1%2) 652. 5. Palade, G E, J exp med 95 (1952) 285. 6. Mackenzie, C G, Mackenzie, J B & Reiss, 0 K, J cell biol 14 (1%2) 269. 7. Skipski. V P & Barclav. M, Methods in ensvmology (ed J M Lowenstein) vol. 14, p. 530. Academic Press, New York (1969). 8. Sheehan, D C & Hrapchak, B B, Theory and practice of histotechnology, p. 128. Mosby, St Louis, MO (1973). Green, H & Kehinde, 0, Cell I(l974) 113. -Ibid 7 (1976) 105. Miiller, U, Ueber den feineren Bau und die Formen der krankhaften Geschwtilste, Berlin (1838). Quoted by Faber (1893). 12. Virchow, R, Virchows arch path hat 14 (1858) 1. 13. Enders, J F & Peebles, T C, Proc sot exp biol med 86 (1954) 277. 14. Marston, R Q, Proc sot exp biol med 98 (1958) 853. 1.5. Okada, Y, Exp cell res 26 (1%2) 98. 16. Papahadiauoulos. D, Mavhew, E, Paste, G, Smith, S & Vail; W J, Biochim biophys acta 323 (1973) 23. 17. Ahkong, Q F, Cramp, F C, Fishe, D, Howell, 3 Tamp&W, Verrinder, M & Lucy, J A, Nature new biol242 (1973) 215. 18. Defendi, V & Stoker, M G P, Nature new bio1242 (1973) 24. 19. Oehlert, W, Seemayer, N & Lauf, P, Beitr path hat 127 (1962) 63. Received September 8, 1976 Accepted December 8, 1976

1. Ivanova, 0 Y & Margolis, L B, Nature 242 (1973) 200. 2. Folch, .I, Lee, M & Sloane Stanley, G H, J biol them 226 (1956) 497.

Exp Cdl Hes 106 (1977)