Cell Attachment to Collagen: The Requirement for Energy ROBERT J. KLEBE Diuision of H u m a n G e n e t i c s , Depccrtment of H u m a n Biological C h e m i s t r y a n d G e n e t i c s , University of Tencis Medzcc7l Brtrnch, Galveston, Texcis 77550

ABSTRACT I t has previously been demonstrated that cell attachment to collagen depends on the presence of a serum-derived cell attachment factor. Two steps in the cell attachment process have been defined. First, the cell attachment factor can bind to collagen in the absence of divalent cations. Secondly, either Ca++or Mg++ are required for cells to attach to the collagen-cell attachment factor complex. A third requirement for cell attachment to collagen is demonstrated here; namely, cellular metabolic energy. A simple method for the assay of ATP in tissue culture cells is presented.

assay is briefly described below. In order to allow the cell attachment factor to bind to collagen, urea-treated collagen-coated plastic petri plates' (Falcon No. 1007) were exposed to various concentrations of serum, ranging from 10 to 0.03%, for 30 minutes prior to the addition of cells. CHO cells were dislodged from a glass surface with 0.25% Bacto-Trypsin (1 :250) (Difco, Detroit, Michigan) and washed twice with medium plus 200 pglml bovine serum albumin (BSA) (Sigma). Depending on the experiment, the medium employed was either Eagle's minimal essential medium (GIBCO) (Eagle, '59) or phosphate-buffered saline (with or without glucose) (Dulbecco and Vogt, '54). 2 X 1 0 . 5 cells were added to the factorized collagen-coated petri plates and incubated at 37°C for one and one-half hours. At the end of this period, the plates were washed with 0.9% NaCl and the cells attached to the collagen were trypsinized and counted METHODS A N D MATERIALS with a n electronic cell counter. The unit of attachment activity is calIn uitro m e t h o d s . Chinese hamster ovary (CHO-LA) cells were cultivated in F-12 me- culated by (a) determining that percent sedium (Ham, '65) supplemented with 4 X rum that attaches 50% of the cells attached amino acids, 2 X vitamins, 10% fetal calf in the control (10% serum points) and (b) serum, and 100 unitslml penicillin and 100 taking the reciprocal of this percent serum 1 pg/ml streptomycin. (unit = v serum M ) (Klebe, '74). Thus, if In these experiments concerning energy utilization, cells were given a complete me- half of the cells, attached in the 10% sedium change at least a n hour prior to use rum control, attach at 2 % serum, the seReceived Nov. 4, '74. Accepted Feb. 3, '75. in order to standardize the initial glucose 1 Collagen should be prepared and the coated petri pool size. plates should be urea-treated as previously described Cell a t t a c h m e n t assay. The assay for (Klebe, '74). Without these precautions, up to 30% of the may attach without serum. Thisbackground is probcell attachment to collagen has previously cells ably due to contamination of rat tail collagen with a rat been described in detail (Klebe, '74). The cell attachment factor.

Mammalian cells attach and spread on a collagen substrate only in the presence of serum-derived cell attachment factor (Klebe, '74). This cell attachment factor has been purified from serum and it has been shown (a) that the factor is a high molecular weight protein, (b) that the factor binds to collagen in the absence of divalent cations or cells, and (c) that either Ca++or Mg++are required for cells to attach to the collagen-cell attachment factor complex (Klebe, '74). It is demonstrated here that cell attachment to collagen requires the utilization of cellular ATP energy. In the course of this investigation, we have developed a fluorimetric ATP assay, suitable for use with tissue culture cells, by adaptations of the standard hexokinase-glucose-6-phosphate dehydrogenase method for ATP (Greengard, '65).

J . CELL.PHYSIOL.,86: 231-236

231

232

ROBERT J . KLEBE

rum contains (or the drug treatment permits cell attachment equivalent to) 0.5 units of cell attachment activity. Simultaneous cell lysis and enzyme inactivation. In the ATP assay, described below, it is essential that the method of cell lysis not result in the loss of ATP by (a) trapping of ATP in cell debris, or (b) enzymatic degradation of ATP. Several methods of cell disruption that are quite satisfactory for large pieces of tissue are not suitable for tissue culture cells. Trichloracetic acid (TCA) and perchloric acid (PCA) treatment of tissue culture cells results in the fixation of cells to glass and plastic surfaces without cell disruption. Since detergents do not disrupt cells after TCA and PCA fixation, TCA and PCA treatments necessitate the removal of cells from a surface with a rubber policeman and homogenization in order to liberate trapped ATP. ATP can easily be physically lost in these procedures that are required after TCA and PCA treatment. Without the use of deproteinizing agents, detergent lysis of cell monolayers does not result in enzyme inactivation and, hence, ATP can be enzymatically degraded. Sodium dodecyl sulfate does lyse cells and inactivate enzymes; however, it also results in ATP break-down. A cell lysis solution has been developed that overcomes the problems indicated above. Rapid cell lysis and simultaneous enzyme inactivation can be achieved with a cell lysis solution consisting of 8 M urea 1% Triton X-100. The procedure for cell lysis is as follows. Cell monolayers in 3 oz glass bottles were washed twice with 5 ml of phosphate-buffered saline (minus glucose) and then 2 ml of the cell lysis solution was added. Cell disruption occurred instantly upon contact with the cell lysis solution. The cell lysate was clarified by centrifugation at 22,000 X g for 15 minutes. Under these conditions, ATP solutions remained stable for a t least six hours without the necessity of boiling the lysate. Up to 0.5 ml of 8 M urea 1% Triton X-100 cell lysate can be used in the 4.8 ml ATP assay without affecting the end-point. Fluorimetric ATP determination. The ATP pool size in mammalian cultured cells has previously been determined by use of the luciferase method (Vlodavsky et al., '73; Anastasia et al., '73; Warner et al., '72; Chapman et al., '71), or a radioactive assay

+

+

employing DNA polymerase (Walters et al., '73). A fluorimetric method is described here that can detect ATP in 1.25 X 105 cells. The fluorimetric method employed in this study is based on the standard hexokinase-glucose-6-phosphate dehydrogenase method for ATP (Greengard, '65) and employs a new method for rapidly stopping cellular metabolism and lysing cells (described above). Two stock solutions are employed in the ATP determination. G-6-PD Solution consists of 30 mg NADP and 0.05 ml glucose6-phosphate dehydrogenase (G-6-PD) (1,980 unitslml) (Sigma G-6PD Type XI from Torula yeast) dissolved in 416 ml of 0.05 M Hepes, pH 7.55. Hexokinase Solu5 ml tion consists of 5 ml of 1 M MgC12 0.05 ml Hexokinase of 0.01 M D-glucose (1,893 unitdml) (Sigma HK Type C-130 from yeast) dissolved in 40 ml of 0.05 R.I Hepes, pH 7.55. G-6-PD and Hexokinase Solutions are aliquoted in 40 and 10 ml quantities, respectively, and frozen a t - 11 "C. The stock solutions remain stable for several months. The ATP assay was performed as follows. First, 0.5 ml of clarified celllysate was added to 3.8 ml of G-6-PD Solution to determine the glucose-6-phosphate concentration. After 15 minutes at room temperature, NADPH was read with an Amico-Bowman spectrofluorimeter at excitation and emission wavelengths of 340 and 460 nm, respectively. In order to assay the ATP concentration, 0.5 ml of Hexokinase Solution was then added; and NADPH was determined, as above, after 45 minutes incubation a t room temperature. The ATP value was constant from 25 to a t least 120 minutes after the addition of the Hexokinase Solution. A standard ATP curve, which ranged from 0.002 to 0.2 p moles ATP plus a buffer blank, was always constructed. ATP levels in the samples were calculated by: (a) multiplying the glucose-6-phosphate

+

+

reading by (4.3) to correct for dilution, (b) (4.8) determining the difference in arbitary fluorescence values between the glucose-6phosphate and ATP readings, (c) subtracting the value of the buffer blank from all differences (from (b)) and (d) comparing the corrected reading (from (c)) with the standard ATP curve. ~

233

REQUIREMENT FOR ENERGY IN CELL ATTACHMENT

The above method gives a linear response with 0.002 to 0.2 p moles of ATP or 0.5 ml of cell lysate containing 1.25 to 25 X 10.5 CHO cells. This assay could potentially read the ATP content of far fewer cells by using smaller fluorimeter cuvettes and could easily be adapted to measure the intracellular pool size of many other metabolites. The assay described here yields a value of 2.6 X 10 - l a moles ATP/CHO cell which is comparable to values ranging from 2 x 10- l.5 to 8.4 X 10-1.5 moles ATP/cell as determined by the luciferase method (Vlodavsky et al., '73; Anastasia et al., '73; Warner et al., '72; Chapman et al., '71). It is important to note that glucose at a final concentration of greater than about 1 0 - 2 M produces an artifact in this assay due to traces of glucose dehydrogenase in G-6-PD which act on glucose to form gluconolactone and NADPH (Greengard, '65). RESULTS

It had previously been demonstrated that cell attachment to collagen requires a high molecular weight serum protein (cell attachment factor), either Ca++or Mg++,Na+, and a buffer to maintain a pH of between 6.0 and 8.5 (Klebe, '74). It was noted that the addition of glucose had a variable effect on the degree of cell attachment to collagen (Klebe, '74). A residual and long-lived pool of intracehlar glucose and ATP can now explain this finding. It is shown below (a) that a significant amount of ATP remains in cells up to four hours after deprivation of a carbon source and (b) that conditions which inhibit ATP synthesis markedly reduce the ability of cells to attach to a collagen substratum. Effect of metabolic inhibitors o n cellular ATP levels. In order to monitor the effect of a carbon source on cell attachment, the ATP pool size was determined under conditions described below. The intracellular level of ATP was determined in the following fashion. About 16 hours prior to an assay, 5 x 1 0 6 cells were planted in 3 oz glass bottles in 7 ml of F-12 medium. The initial glucose pool size was standardized by giving cells a complete medium change at least one hour prior to any treatment. An experiment was initiated by washing the cell sheet three times with phosphate buffered saline (minus glucose) and adding a test medium. The incubation

period was terminated by washing the cell sheet twice with phosphate buffered saline (minus glucose), adding 2 ml of cell lysis solution, and preparing the cell extract for fluorimetric ATP determination as described in the MFTHODS A N D MATERIALS section. After glucose deprivation, the ATP pool in CHO cells falls slowly and considerable amounts of ATP remain after four hours (table 1). Hence, removal of a carbon source would not be expected to affect an ATP dependent phenomenon for several hours. Therefore, conditions were devised that resulted in rapid inhibition of ATP synthesis. Treatment of cells with several metabolic inhibitors in the presence of 2 mg/ml glucose had little effect on ATP levels (except for the irreversible sulfhyl reagent, N-ethylmaleimide) (table 2). In contrast, exposure to cells to these metabolic inhibitors in the absence of glucose results in a decrease in ATP pool size within 15 minutes and a decline in ATP levels to values between 7 and 19% of the control value in one hour (table 2). Vlodavsky et al. ('73) and Warner and Perdue ('74) have reported similar residual levels of ATP after drug treatment with the use of the luciferase assay. These residual levels of ATP are possibly due to the availability of unblocked pathways €or ATP synthesis. Effect of metabolic inhibitors o n cell attachment to collagen. The energy requirement for the establishment of cell attachment to collagen was investigated as follows. Cell attachment assays were performed on collagen using cells washed twice in PBS-glucose 200 pg/ml BSA as previ-

+

TABLE 1

ATP pool size a f t e r remono1 of qlucosr Time after removal of glucose (hr)

Moles ATPjcell

to

0.25 0.5 1

2 4 ~~

~

~~

Monolayers of 5 X 106 CHO cells were fed with f'resh F-12 medium one hour prior to the experiment. The assay period was initiated hy washing the monolayers three times with PBS ( -glucose) and terminated by washing twice with PBS ( - glucose) and adding cell lysis solution. ATP levels were determined as described in the METHODS A N D MATERIALS section.

234

ROBERT J . KLEBE TABLE 2

ATP pool s i z e ciJter trecitmnit with metciboltc mhzbitors ATP pool size 15 inin

Metabolic inhibitor Inhibitor

+ Glucose

Site of action

Control N a Azide, 3 X 10-8 M DNP, 10 4 M NEM, 1 0 - 4 M Oligomycin, 5 X 1 0 - 7

-

100 ‘G 99 99 43 85

Electron t r a n s p o r t Uncoupler Glycolysis i n h i b i t o r Energy transfer

M

1

1 hr

- Glucose

+ Glucose

104.59: 20 47

1185 18 19 11 7

1005: 104

118 12 71

32 78

- Glucose

5 X 106 CHO cells were planted i n 3 0 2 . Sani-glas bottles 16 hours before the experiment. One hour prior to the addition of drugs, the cells were re-fed with 7 ml of F-12 medium. Incubations were done with PBS ( t 2 mglml glucose) for 15 minutes and one hour and terminated by washing the cell sheets twice with PBS - glucose and lysing cells with 8iM urea l r r Triton X-100 (cell lysis solution) DNP: 2,4-Dinitrophenol; NEM: N-ethylmaleimide looc; ATP = 2.6 X 10- moleicell.

+

ously described (Klebe, ’74) except that serum treated collagenized plates were chilled for 15 minutes at 4°C prior to the addition of cells. The use of chilled plates allows cells time to respond to the drug, rather than to their to condition. Exposure of cells to metabolic inhibitors results in a marked decrease in cell attachment to collagen and this effect is reversed by 2 mg/ml D-glucose (except for the irreversible sulfhydryl reagent, N-ethylmaleimide) (table 3). We TABLE 3

Cell cittcichment to collcigen cifter trecitment w i t h metctbolzc inhibitors Cell attachment Inhibitor

Control N a Azide, 3 X 10-3 M DNP, 1 0 - 4 M NEM, 1 0 - 4 M Oligomycin, 5 X 1 0 - 7

+ Glucose

- Glucose

TABLE 4

100“r 65

5526% 1 8 11 (4 2 ) 1

D e t a c h m e n t of cells cifter trecitment w i t h metabolic inhibitors

126 ( 9 3 2 ) 1 M

have noted partial reversibility of drug effects on cell attachment at glucose concentrations as low as 20 pglml. Trypsinized monolayer cells or cells grown in suspension respond to 2,4-dinitrophenol in a quantitatively similar fashion. The energy requirement for the maintenance of cell attachment to collagen was investigated as described below. Cells were allowed to attach to collagen for one and one-half hours in the absence of glucose at which time metabolic inhibitors were added. Exposure of cells to metabolic inhibitors results in detachment of cells within one and one-half hours (table 4). Most of the cells that had attached in one and one-half hours to collagen in the ab-

85

8

Stock CHO cultures were fed with fresh medium one hour prior to the beginning of the assay. Trypsinized cells were washed twice with PBS ( -glucose). 2 X 105 cells were added to chilled petri plates containing PBS ( -C 2 mg/ml glucose) and dialyzed serum (10, 3, 1, 0.3, 0.1, 0.03,and 09; ). The plates were incubated for 1.5 hours a t 37’C and the number of attached cells were counted. The data is expressed a s the percent of the number of units of attachment activity in the +glucose control. DNP: 2,4-Dinitrophenol; NEM: N-ethylmaleimide. It should be noted t h a t the -glucose control value varies &om experiment to experiment. This is most probably due to differences i n the metabolic state of cultures which were harvested at different degrees of confluency. 2 In order to control for the effect of trypsinization, CHO cells were grown i n suspension, harvested by centrifugation, and assayed as described in the METHODS AND MATERIALS section.

Cell attachment Metabolic inhibitor

Control N a Azide, 3x M DNP, 1 0 - 4 M NEM, 1 0 - 4 M Oligomycin, 5 x 10-7 M

41

0.5hr

1.5 hr

3 hr

100%

100%

93

19

-

106

64

8

25 70

I1

3

1

1

-

33

7

3

CHO cells were washed in PBS - glucose a n d 2 X 105 cells were allowed to attach to collagen i n PBS-glucose 0.03, in the presence of dialyzed serum (10, 3, 1, 0.3,0.1, a n d 0% ), After 1.5 hours (t,) inhibitors were added and the number of cells remaining attached were assayed a t 0.5, 1.5, and 3 hours. The d a t a is expressed as the percent of the number of units of attachment activity in the t,, control. DNP: 2,4-Dinitrophenol; NEM: N-ethylmaleimide.

REQUIREMENT FOR ENERGY IN CELL ATTACHMENT TABLE 5

Temperciturr d e p e n d e n c e of cell u t t c i c h m e n t Temperature ('C)

Cell attachment u n i t s

40 12" 23 30 36.5'

0.09 0.43 0.67 1.25 1 .oo

~~

~~

Collagen-coated petri plates were exposed to serum (10,3, 1, 0 . 3 , 0.1,0 . 0 3 a n d O r ; 1 for 30 minutes at 37'C. The factorized plates were then chilled to 4'C prior to addition of 2 X 103 CHO cells. The plates were then incubated at the temperatures indicated for 1.5 hours and assayed for cell attachment a s described i n the METHODS A N D SECTION.

DNP: 2,4-Dinitrophenol; NEM: N-ethylmaleimide.

sence of glucose detach after an additional three hours deprivation of glucose (table 4) without the addition of metabolic inhibitors. Temperature dependence of cell attachment. Cell attachment to collagen is temperature dependent as is shown in table 5. Evidence against microtubular involuem e n t in cell attachment. Cell deformation has been shown to be dependent on energyrequiring microtubular assembly (Piatigorsky et al., '72; Weisenberg, '72). Thus, microtubular assembly could be the energy requiring step involved in cell attachment if a cell must spread itself in order to establish and maintain its hold on collagen. Microtubules do not appear to be involved in cell attachment since concentrations as high as lo-" M colchicine and 10-4 M vinblastine sulfate are not inhibitory. DISCUSSION

The evidence presented here indicates that metabolic energy is required for CHO cells to initiate and maintain their attachment to a collagen substratum. Three lines of evidence can be sited: (A) Several agents which block the synthesis of ATP also inhibit cell attachment; and glucose reverses these effects. (B) Low temperature treatment results in a 90% inhibition of cell attachment to collagen. (C) Metabolically dead fibroblasts do not attach to a substrate. (Cell attachment has long been a generally accepted criterion for cell viability and metabolic integrity.) Grinnel and Srere ('71) have previously shown that several sulfyhdyl blocking agents, including N-ethylmaleimide, inhibit rat hepatoma cell (HTC) attachment to

235

polystyrene surfaces. It has been demonstrated here that N-ethylmaleimide irreversibly inhibits both cell attachment to collagen and ATP synthesis. In addition, it has been found that N-ethylmaleimide does not effect the binding of the cell attachment factor to collagen since only direct treatment of cells with N-ethylmaleimide inhibits cell attachment. The role of cellular metabolism in cell attachment to collagen is not clear. The possibility of the involvement of energy requiring microtubular assembly (Weisenberg, '72) in the ability of a cell to attach to collagen seems to be ruled out since cell attachment is not inhibited by high concentrations of colchicine and vinblastine sulfate. In contrast, low concentrations of colchicine immediately block the related phenomenon of cell spreading (Piatigorsky, '72). From the evidence presented, three steps in cell attachment can be defined. First, the cell attachment factor must bind to collagen. This step does not require divalent cations or cells (Klebe, '74). Secondly, either Ca++or Mg++must be present for a cell to attach to the collagen-cell attachment factor complex (Klebe, '74). Thirdly, cellular energy must be exerted for a cell to initiate and maintain its attachment to a collagen substratum. ACKNOWLEDGMENTS

The author thanks Mrs. Ann Davis for her excellent technical assistance. This work was supported by a grant from the National Institutes of Health (GM2 1433). LITERATURE CITED Anastasia, J . V . , and R. L. McCarl 1973 Effects of cortisol on cultured rat heart cells. Lipase activity, fatty acid oxidation, glycogen metabolism, and ATP levels as related to the beating phenomenon. J. Cell Biol., 57: 109-116. Chapman, J. D., R. G. Webb and J. Borsa 1971 ATP pool levels in synchronously growing hamster cells. J. Cell Biol., 49: 229-233. Dulbecco, R., and M. Vogt 1954 Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med., 99: 167-182. Eagle, H . 1959 Amino acid metabolism in mammalian cell cultures. Science, 130: 432-437. Greengard, P. 1965 Adenosine-51-Triphosphate determination by fluorimetry. In: Methods of Enzymatic Analysis. H . Bergmeyer, ed. Academic Press, New York., pp. 551-558. Grinnell, F., and P. A . Srere 1971 Inhibition of cellular adhesiveness by sulfhydryl blocking agents. J . Cell Physiol., 78: 153-157.

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Ham, R. G. 1965 Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Nat. Acad. Sci., (U.S.A.),53: 288-293. Klebe, R. J . 1974 Isolation of a collagen dependent cell attachment factor. Nature, 250: 248-251. Piatigorsky, J., H. D. Webster and M. Wollberg 1972 Cell elongation in the cultured embryonic chick lens epithelium with and without protein synthesis. Involvement of microtubules. J. Cell Biol., 55: 8%92. Vlodavsky, I . M . Inbar a n d L. Sacks 1973 Membrane changes and adenosine triphosphate con-

tent in normal and malignant transformed cells. Proc. Nat. Acad. Sci., (U.S.A.), 70: 178&1784. Walters, R. A , , R. A . Tobey and R. L. Ratliff 1973 Cell-cycle-dependent variations of deoxyribonucleoside triphosphate pools in Chinese hamster cells. Biochim. Biophys. Acta, 319: 33C347. Warner, D. A , , a n d J. F. Perdue 1972 Cytochalasin B and the adenosine triphosphate content of treated fibroblasts. J. Cell Biol., 55: 242-244. Weisenberg, R. C. 1972 Microtubule formation in vitro i n solutions containing low calcium concentrations. Science, 177: 1104-1105.