Factors regulating the multiplication of animal cells in culture

Printed in Sweden Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved ISSN 00144827

Experimental

FACTORS

Cell Research 107 (1977) 159-167

REGULATING

OF ANIMAL J. HASSELL’ Department

of Microbiology,

THE MULTIPLICATION

CELLS

IN CULTURE

and D. L. ENGELHARDT

College of Physicians and Surgeons, Columbia New York, NY 10032, USA

University,

SUMMARY The multiplication of Vero cells in vitro as measured by population doubling time and final saturation density is regulated by the serum concentration in the medium and not by the substratum area available for cell growth. Vero cells at high density deplete the medium of a serum component(s) essential for continued growth. They also display a density dependent response to the removal of serum from the growth medium. At high densities, they continue multiplying longer than at low densities. No difference is noted in these uronerties among the enithelial cell tvoes: CV-1, BSC-1 and 2” African green monkey kidney (AGM-K) cells, nor-among the fibroblastcell tvues: 3T3 and SVlOl. These data are consistent with the model that the growth of most cultured ceils is controlled by medium components especially serum growth factors and that no difference exists in this respect between epithelial cells and fibroblasts.

Studies with the mouse embryo fibroblast fibroblast is caused by a limitation of mecell line 3T3 have demonstrated the pivotal dium components, especially serum. role played by medium components, espeThe multiplication of other fibroblastic cially serum growth factors, in regulating cells is also regulated by serum. These incell multiplication (see [12] for a recent re- clude a baby hamster kidney (BHK) cell view). The final saturation density as well line [2] and primary cultures of chick emas the rate of cell population doubling vary bryo flbroblasts [ 181. with the extracellular serum concentration. Much less is known about the factors that At relatively high serum concentrations regulate the multiplication of epithelial these cells have shorter doubling times cells, though the suggestion has been made and attain higher saturation densities than that cell multiplication is controlled by difat low serum concentrations [14]. Other ferent mechanisms in fibroblastic and epistudies have demonstrated that under nor- thelial cell cultures [3, 4, 221. This suggesmal culture conditions the maximum cell tion is based on two lines of evidence. (1) density of stationary phase 3T3 cells is re- If a wound is made in a density-inhibited stricted by depletion of medium compo- culture of fibroblastic cells such as 3T3, the nents [4,13]. These groups have concluded remaining cells migrate into the denuded that density dependent growth inhibition of area, initiate DNA synthesis and eventually divide only if serum factors are added to the ’ Present address: Cold Spring Harbor Laboratory, medium. Epithelial cells, however, do not P.O. Box 100, Cold Spring Harbor, NY 11724, USA. 1 I-771818

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Fig. I. Abscissa: substratum surface area (cm2); ordinate: saturation density (cellsx 10-5/cm2). The effect of substratum surface area on the saturation density of Vero cells. Vero cells were inoculated onto plastic dishes of varying surface areas at 5xloJ cells/dish in 6 ml of growth medium. Cell number was then determined in duplicate every second day until the saturation density on dishes of varying area had remained constant after two consecutive determinations.

contamination using the procedure of Todaro et al. [20]. These cell lines were cloned in this laboratory and subcultured by transfer of lo4 cells to 100 mm diameter Falcon plastic dishes every 4-6 days. Cells were not allowed to attain confluence before being passaged. African green monkey kidney (AGMK) cells were purchased from Gibco (Grand Island, N.Y.) and from Microbiological Associates (Bethesda, Md) as a cell suspension. They were inoculated onto 100 mm Falcon plastic dishes, and the medium changed 24 h after plating. When the cells had attained confluence, they were subcultured and used directly for cell growth measurements. These cells are referred to as AGMK secondary cultures. Vero cells, the line used most extensively in this study, were anchorage-dependent in growth in that thev did not arow in agar or methocel suspension (Has&l, unpublished data). Cells were identified as euithelial (eoithelial-like) or tibroblastic (fibroblast-likej accord& to the criteria of Federoff [7].

Cell growth measurements In general, cells were seeded onto Falcon plastic dishes (surface area of 19.6 cm*) in 4 ml of DME medium supplemented with calf serum (concentrations are specified in the figure captions). The medium was changed every day thereafter. Duplicate cultures were used to determine cell number as follows. The cell

require exogenous serum to initiate DNA synthesis or multiply in a wound [3], (but see [12]). (2) The growth of fibroblasts in dense culture is restricted because of medium depletion, while the surface area available for cell attachment limits the multiplication of epithelial cells [4]. Since most human cancers are of epithelial origin [ 111, we have begun to study the growth properties of epithelial cells. For these studies we have used African green monkey kidney (AGMK) cells and lines derived from these cells, especially Vero cells, as model systems. MATERIALS

AND METHODS

Cell maintenance The cells used in this study were grown in high glucose (4.5 g/l) Dulbecco’s modified medium (DME) supplemented with 10% (v/v) calf serum (growth medium) in a humidified 5 % CO* atmosphere. All cell lines except BALB/c 3T3 (obtained from Dr G. Todaro) were purchased from the American Type Culture Collection. They were routinely screened for mycoplasma ExptlCellRes

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Fig. 2. Abscissa: time (days); ordinate: cellsx10-4/ cm*. Growth curve of Vero cells with O-O, daily medium changes, or O-O, without a medium change after plating. Cultures of Vero cells were seeded onto 19.6 cm* Falcon plastic dishes containing 4 ml of growth medium on day 0. On day 7 (arrow) the medium was changed for half the cultures which had not experienced a change of medium, and A-A, changed daily thereafter. Cell count was determined in duplicate and the average plotted.

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RESULTS Growth limitation of Vero cells To investigate if medium components or substratum surface area limited the growth of Vero cells at high cell density, we performed the following experiment originally described by Dulbecco & Elkington [4]. Vero cells were seeded at the same initial cell number in culture dishes of different surface areas, and 6 ml of growth medium placed on all the dishes. The saturation density of all the cultures was then determined after cell division had ceased. The results, presented in fig. 1, show that the saturation Figs 3-5. Each value is the average of two determina- density did not depend on the surface area tions. Fig. 3. Abscissa: time (days); ordinate: cellsx 10-Y available for cell adherence. The final cell cm2. number on all but the smallest dish was apThe growth of Vero cells in medium of different serum concentrations. Vero cells were seeded onto 19.6 proximately the same (6.5-7.5~ lo6 cells/ cm* Falcon plastic dishes in 4 ml of growth medium; dish); this corresponded to 1. l-l .3 x lo6 24 h later the medium was changed to medium con- cells/ml of growth medium. Only at extrataining O-0,0.5; A-A,2.O;e-O,S.O;0-0, 10% (v/v) calf serum. The medium was then changed daily ordinarily high cell densities did the subthereafter. stratum surface area apparently limit cell

‘I- I

layer was rinsed twice with 4 ml portion of PBS (0.01 M sodium phosphate, pH 7.4, 0.15 M NaCl), and the cells removed by digestion in 1 ml of TE (0.05 % trypsin, 0.005 M EDTA in PBS) at 37°C. The trypsin was then blocked by adding an aliquot of 10% calf serum in DME, and a portion of the resultant cell suspension used for counting in a hemocytometer chamber. At least 250 cells, and in most cases 500-l 000 cells were counted in duplicate for each sample. The population doubling time was calculated after 2-3 generations during the exponential phase of growth. Saturation densities were attained when the number of cells/cmz of substratum surface area was maintained for at least 3 consecutive days in culture with daily medium change.

Cell division in serum-free medium To deprive cells of serum the growth medium was removed, and the cell layer rinsed twice with 5 ml volumes of serum-free DME. Four ml of serum-free DME was then added to each dish, and the increase in cell number was measured at the time when the control cultures (cells dividing in growth medium) experienced a 100% increase in cell number. This time interval corresponded to the normal population doubling time of cells dividing in growth medium. For Vero, CV-1 and AGMK secondary cultures this was approx. 18-20 h, while for 3T3 and SVlOl (an SV40-transformed 3T3 cell line) this was 12-14 h.

Fig. 4. Abscissa: serum cont. (% v/v); ordinate: (left) saturation density (cellsx 10-s/cm*) (0-O); (right) generation time (hours) (O-O). The saturation density and generation time of Vero cells growing in medium containing different serum concentrations. Both were determined as described in Materials and Methods.

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Table 1. Saturation density of different cell types in I % and 10 % calf serum Saturation density (cellsx lO+/cm’) Cell type

1% calf serum

10% calf serum

3T3 AGMK Vero cv-1 BSC-1

0.1 1.0 2.5 1.3 1.8

0.7 4.0 9.0 4.5 7.6

Cells were seeded at 0.5-2~10’ cells/cm* in Falcon plastic dishes (19.6 cm*) in 4 ml of medium containing 1% (v/v) or 10% (v/v) calf serum. The medium was changed daily. Cell number was determined in duplicate every two days. The saturation density is the cell number/cm* which the cells maintained for 4 consecutive days in culture.

cells grown without medium change remained viable in the stationary phase. They displayed no loss in their efficiency of cloning, and they excluded trypan blue. Their mitotic index dropped to less than onetenth that of growing cells, and they accumulated in the Gl (or GO) phase of the division cycle [5,6]. Thus they are in a normal stationary phase, and the attainment of this state is regulated by factors in the medium. The multiplication of fibroblasts is controlled to a large extent by the medium serum concentration [2, 7, 141.We measured whether serum concentration regulates the growth of Vero cells [see 8, 9, lo]. To do this the population doubling time and saturation density were calculated from growth curves of Vero cell cultures with medium containing 0.5, 2.0, 5.0 or 10% (v/ v) calf serum. The results shown in fig. 3 revealed the following. For the first two days

division (fig. 1). Thus the substratum surface area does not limit the multiplication of Vero cells under normal culture conditions. A second technique to distinguish whether medium components or substratum surface area limits Vero cell growth is to determine the effect of either daily medium change or no medium change on final sa- Table 2. The serum concentration of condituration density and population doubling tioned medium time. The results of these determinations Saturation Serum are shown in fig. 2. The frequency with density assayed (cells/cm*) which the medium was changed did not al- Medium (%) ter the population doubling time of 18 h for Growth medium 8.7x 105 >5 Vero cells. The saturation density, how- Control growth medium 7.6X 105 >5 3.5x 105 1.5 ever, depended markedly on the number of Conditioned medium of conditioned and medium changes. Daily replenishment of Mixture control growth medium 7.2x l(r >5 medium resulted in Vero cells attaining a Vero cells were seeded onto Falcon tissue culture saturation density of 9x lo5 cells/cm2, while flasks (75 cm%)in 25 ml of growth medium at 5x 10’ if the medium was not changed after initial cells/cmZ and allowed to grow to a saturation density of 3-4x 105cells/cm*. The medium, referred to here as plating the cells grew to only half this den- conditioned medium, was collected from six flasks, sity. Moreover, if daily medium change was dialysed against growth medium for 2 days at 4”C, and then assayed for its growth-promoting capacity by albegun on cultures that had no medium lowing Vero cells to divide in this medium with daily change and had stopped dividing, these cells changes, and measuring their saturation density. The capacity of conditioned medium resumed growth until they had reached the growth-promoting was then compared with fig. 4. Control growth medium same saturation density as cultures which refers to growth medium which was subjected to exactly the same experimental manipulations as depleted had experienced daily medium changes medium (exposure to plastic flasks, 37” for 6 days, etc.) throughout the growth period (fig. 2). These but not exposed to cells. Exptl Cell Res 107 (1977)

Factors regulating animal cell multiplication

Fig. 5. Abscissa: time (days); ordinate: cellsx10-4/ cmp. The growth response of Vero cells to serum deprivation. Vero cells were inoculated onto 19.6 cm* Falcon plastic dishes in 4 ml of growth medium (O-O). On day 2 some of the cultures were deprived of serum (O-O), and the medium changed for the remainder (controls). On day 5, growth medium was added to half of the serum-deprived cultures (A-A), and the medium changed for all the other cultures as well.

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4 illustrates more clearly this relationship between the population doubling time, the saturation density and serum concentration for Vero cells. The data shown in fig. 4, while illustrating the dependence of Vero cell multiplication on serum concentration, also shows that these cells have a relatively low serum requirement for cell division. To determine if this was an idiosyncrasy of this cell line or a general characteristic of monkey cell lines or strains, we compared the saturation density of Vero, CV-1, AGMK secondary cultures, BSC-1 and 3T3 cells at 1.O% (v/v) serum concentrations. The tabulated results (table 1) show that 3T3 cell cultures have a more stringent serum requirement for multiplication than the monkey cells tested. The AGMK cell lines and secondary cultures all display a serum requirement for

10 0P 6.0 4.0 20 ‘.I3 80 0rfn40 120 160

in culture after the change to different serum concentrations the initial growth rate remained unchanged in all the cultures. (The cells were seeded at the same cell density in 10% (v/v) calf serum containing medium, the medium changed 24 h later to DME containing different concentrations of calf serum, and then daily thereafter.) When the cell density reached 6-7~ lo4 cells/cm2, the rate of growth changed in some of the cultures dependent upon the serum concentration of the medium. In three separate repetitions of this experiment, it was ob- Fig. 6. Abscissa: % increase in cell count in serum-free ordinate: cellsx 10~*/cm2. O-O, Expt 1; served that this decrease in growth rate oc- medium; O-O, expt 2; A-A, expt 3. curred at a characteristic cell density (0.6The percentage increase in cell number of Vero cells serum-free medium after 18 h of serum starvation. 1.0~ lo5 cells/cm2) for 0.5 and 2.0% serum in Vero cells were grown as described in the caption to concentrations, rather than after a defined fig. 5, and deprived of serum at different densities durexponential growth. The percentage increase in period of time in culture. Furthermore, as ing cell number occurring in the serum-deprived cultures is illustrated in fig. 3, the cells that divide was then calculated as described in Materials and This graph is the composite of three separate more slowly in low serum (0.5 and 2 %) also Methods. experiments. Each point is the average of duplicate attain a lower final saturation density. Fig. measurements of cell number.

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Table 3. The eflect of serum-free medium volume on cell division Cell line Vero

Medium vol (ml)

Increase in cell number in serum-free medium (%)

: 8 10 18

70 56 38 31 27

ing capacity equal to that of the growth medium alone. We conclude from this that Vero cells deplete the medium of a serum component(s), and that this serum depletion may restrict the growth of these cells at high density [see 191. Cell multiplication in serum-free medium

To further characterize the requirement by Vero cells for serum for multiplication, the Vero cells at a density of from 3-5~ 10 cells/cm* (100 mm 0 plastic dishes) were deprived of serum, and the capacity of these cells to multiply in the abmedium replenished with different volumes of serum- sence of serum was assayed. This was done free medium. Cell number was then determined exactly as described in the caption to fig. 5. by depriving them of serum for varying periods and measuring the percentage increase in cell number at the end of these growth, but these differ quantitatively from periods. There was a 50% increase in cell one another (table 1). number after 18 h (the population doubling Serum could be limiting for cell multiplitime of the control cultures) in the absence cation at high cell densities either because of serum compared to a 100% increase in of the accumulation of a cell-specified mul- the control cultures (fig. 5). In the contiplication inhibitor or due to the depletion tinued absence of serum the cells eventually of some serum component(s) essential for ceased dividing, and subsequently (after 4 multiplication [ 1, 13, 171.To distinguish be- days in the continued absence of serum) a tween these alternatives Vero cells were al- decrease in cell number occurred (fig. 5). If lowed to reach a stable saturation density serum-containing medium was returned to without medium changes. The medium the non-dividing serum-starved cultures, (conditioned medium) was removed and cell division recommenced after a lag of dialysed against growth medium, and the about one day (fig. 5). saturation density to which Vero cells grew In subsequent repetitions of this experiin this dialysed conditioned medium was ment it was noted that the cell density at the measured. Table 2 shows that depleted me- time serum was removed markedly indium had a growth-promoting capacity fluenced their subsequent growth response. comparable to a serum concentration of When these cells were deprived of serum at 1.5 % (extrapolated from fig. 4), while fresh 1.4x IO4 cells/cm2, there was a 39% inDME containing 10% calf serum (growth crease in cell number after 18 h, while when medium), or growth medium which had they were deprived of serum at 2.2~10~ been exposed to the same experimental cells/cm2 there was a 160% increase in cell manipulations as depleted medium but not number over the same time period (fig. 6). exposed to cells had a growth-promoting The control cultures experienced a 100% capacity comparable to a serum concentra- increase in cell number over the same time tion of greater than 5 %. period (fig. 6). Moreover, at high cell densiWhen this conditioned medium was ties (1 X 10J cells/cm2) cell division was mixed in equal parts with growth medium sustained for a longer period of time than at the resultant medium had a growth-promotlow cell densities (1 x lo4 cells/cm”) in seExptlCellRes

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Factors regulating

rum-free medium (data not reported). Thus the capacity of these cells to divide in serum-free medium is density dependent. 2” AGMK cultures, 3T6 cells and SV40-transformed 3T3 cells also continue multiplying for a longer period when deprived of serum at high cell densities when compared with low densities. At high densities they showed lOO-120% increase in cell number depending on the cell type while at low density they showed 10-15% increase. Thus the capacity of these cells to divide in serum-free medium is also density dependent. In view of the report that some cell types could multiply in serum-free medium [ 161it is interesting to note that little difference was observed in this work in the capacity of cells plated at a low density to divide in serum-free medium. This assay was performed on a wide variety of cells. For example, in an experiment where cells were plated between 5 x 103/cm2and 1x 104/cm2, several cell types showed close to similar increases in cell number. They were 3T3 cells (up 12%), 3T6 (up 13%), SV40-transformed 3T3 (up 13%), flat-revertant SV40transformed 3T3 (a gift of Dr Robert Pollack) (up 15%), B77 virus-transformed A3 1 clone of BALB/c 3T3 cells (a gift of Dr George Todaro) (up 15%), Vero cells (up 39 %), CV-1 cells (up 25 %), BSC-1 cells (up 21%) and 2” AGMK cells (up 15%). Thus none of these cells multiplied to a significant extent in serum-free medium. This density dependent multiplication in serum-free medium might be a consequence of cell density per se (i.e., cell-cell interaction) or due to some factor released into the medium from the monolayer. To distinguish between these alternatives, several culture dishes of Vero cells were deprived of serum at the same cell density, and the volume of serum-free medium added to the dishes was varied (in our previous experiments the

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volume of serum-free medium/dish remained constant). We then measured the percentage increase in cell number after 18 h. Control cultures experienced a 100% increase in cell number over these periods of time regardless of the medium volume. The results (table 3) show that at lower medium volumes more cells divided than at higher medium volumes. This suggests that the rate of cell division is being regulated by the concentration of some component released from the monolayer into the medium, such that, with larger volumes of serum-free medium, the concentration of this component (or these components) is decreased with a consequent decrease in proliferation rate. We have obtained qualitatively similar results with AGMK cultures, 3T6 cells and SV40-transformed 3T3 cells (data not shown). DISCUSSION We examined the growth properties of Vero cells and found that like many other mammalian and avian cells their multiplication is regulated by factors in serum. The saturation density reached when Vero cells were allowed to divide without medium change is not a consequence of a limitation of substratum surface area, or due to cell-cell contacts but is a consequence of the limitation of some medium component. This is indicated since cells growing with daily medium changes reached saturation densities two-fold higher than cultures that did not experience daily medium changes. Also when the substratum surface area for cell attachment was varied, but the growth medium volume held constant, the final number of cells on dishes of varying surface areas was the same for Vero cells (fig. 1). Furthermore, since Vero cells become confluent (extensive cell-cell contact) at 1X lo5 Esptl Cd Res 107 (1977)

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cells/cm2, but do not cease dividing until they reach nine times this density, it is unlikely that an inhibitory effect of contacts among cells blocks cell division at high density. The saturation density and population doubling time of Vero cells can be varied by controlling the concentration of serum in the medium (figs 3 and 4). At 0.5 and 2% (v/v) serum the final saturation density was directly related to the serum concentration, while above these serum concentrations (5 and 10%) the saturation density was less dependent on the serum concentration of the growth medium. As is shown in table 2 growth medium conditioned by Vero cells has had a growth-promoting serum component largely depleted, and cells grown in this depleted medium fail to grow at high cell densities. Also the growth rate and saturation density of AGMK, CV-1 and BSC1 cells varies with the concentration of extracellular serum (table 1; Hassell, unpublished data) in much the same manner as that recorded for Vero cells (fig. 4) and AGMK cells also deplete serum of an essential component(s) for growth (Engelhardt & Mao, unpublished data). These experiments are consistent with the model that the concentration of serum factor(s) regulates the saturation density and population doubling time of epithelial cells derived from monkey kidney. Vero cells have a relatively low serum requirement for growth control when compared to 3T3 mouse embryo fibroblasts. The same is true for AGMK secondary cultures, CV-1 cells and BSC-1 cells (table 2). Since Vero cells were established in medium containing 2 % (v/v) calf serum [23] it is possible that only those cells capable of dividing at this serum concentration were selected. 3T3 cells on the other hand were established in medium containing 10% (v/v) ExprlCellRes

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calf serum [2 11,and this may be reflected in their increased requirement for serum for cell division. However, the fact that all the monkey cells had a lower serum requirement than 3T3 suggests that this is a general property of this type of cells. Finally the multiplication of Vero cells (fig. 6) and AGMK cells (unpublished data) as well as 3T6 and SV40-transformed 3T3 cells (unpublished data) in serum-free medium is markedly dependent on the cell density at which serum is removed from the culture fluid. Serum deprivation at high cell densities had less effect on the subsequent rate of cell proliferation than at low cell densities. These experiments suggest that Vero cells and AGMK cells may either secrete growth factors into the medium, or that it may be technically difficult to remove serum growth factors by washing, especially when the cell density is high. This fact coupled with the observation that AGMK cell lines have a low serum requirement for multiplication (table 2) may explain why epithelial cells apparently do not have a serum requirement for DNA synthesis in a wound [3]. They may also explain the reported association of the capacity for cell division in serum-free (or low serum) medium with the transformed phenotype [14, 15, 161in that transformed cells are routinely handled at higher densities than non-transformed cells. Experiments by Dulbecco & Elkington [4] similar to those recorded in fig. 2 indicated that the multiplication of BSC-1 and CV-1 cells was primarily restricted by the substratum surface area though medium limitation was not ruled out at higher cell densities. We have shown, using many independent measures, that medium components, principally serum, regulate the multiplication of Vero cells, and AGMK secondary cultures. The reasons for the discrep-

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ancy between these data are not clear. We 5. Engelhardt, D L & Mao, J-h, J cell physiol 90 (1977) 307. cannot rule out the possibility that our me- 6. Engelhardt, D L & Samoski, J, J cell physiol 86 (1975) 15. dium or sera are different, or that they were 7. Federoff, S, J natl cancer inst 38 (1967) 607. depleted of critical growth factors faster in 8. Hassell, J A, Colby, C & Romano, A H, J cell physio186 (1975) 37. our experiments. 9. Hassell, J A & Engelhardt, D L, Biochim biophys In summary we have been unable to find acta 324 (1973) 545. any qualitative differences in the basic 10. - Biochemistry 15 (1976) 1375. 11. Higginson, J & Muir, C S, Cancer medicine (ed mechanisms which control multiplication J F Holland & E Frei III) p. 241. Lea & Febiger, Philadelphia, Pa (1973). of certain fibroblastic and epithelial cells. 12. Halley, R W, Nature 258 (1975) 487. Quantitatively different requirements for 13,Ho 11 ey, R W & Kieman, J A, Proc natl acad sci us 60 (1968) 300. serum growth factors have been found, but 14. - Growth control in cell cultures (ed G E W these are likely to reflect individual cell Wolstenholme & J Knight) p. 3. Churchill Livingstone, Edinburgh (1971). strain or line differences rather than general 15. Martin, R G & Stein, S, Proc natl acad sci US differences, These data are consistent with 73 (1976) 1655. the postulate that the growth of most cul- 16. Pitt‘s, J D, Growth control in cell culture (ed G E W Wolstenholme & J Knight) p. 261. Churchill Livtured cells is controlled by medium compoingstone, Edinburgh (1971). 17. Roehm, C & Lipton, A, Nature new biol245 (1973) nents, especially serum growth factors. 115. This research was supported by grant BMS-74-02201A01 from the NSF.

REFERENCES 1. Bullough, W S, Nature 229 (1971) 608. 2. Clarke, G D & Stoker, M G P, Growth control in cell culture (ed G E W Wolstenholme 8z J Knight) p. 17. Churchill Livingstone, Edinburgh (1971). 3. Dulbecco, R, Nature 227 (1970) 802. 4. Dulbecco, R & Elkington, J, Nature 246 (1973) 197.

18. Rubin, H, Growth control in cell cultures (ed G E W Wolstenholme & J Knight) p. 127. Churchill Livingstone, Edinburnh (1971). 19. Stoker, M G P, Nat& 246 (1973) 200. 20. Todaro, G J, Aaronson, S A & Rands, E, Exp cell res 65 (1971) 256. 21. Todaro, G J & Green, H, J cell biol 17 (1%3) 299. 22. Yainiguchi, N &Weinstein, I B, Proc natl acad sci US 72 (1975) 214. 23. Yasumura, Y & Kawakita, Y, Nippon Rinscho 21 (1%3) 1209. Received September 15, 1976 Accepted January 27, 1977