Establishment and Transformation Diminish the Ability of Fibroblasts to

Establishment and Transformation Diminish the Ability of Fibroblasts to

Published Online: 1 October, 1980 | Supp Info: Downloaded from on March 24, 2018 Establishment an...

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Published Online: 1 October, 1980 | Supp Info: Downloaded from on March 24, 2018

Establishment and Transformation Diminish the Ability of Fibroblasts to Contract a Native Collagen Gel BETTIE M. STEINBERG, KATY SMITH, MARY COLOZZO, and ROBERT POLLACK Department of Biological Sciences, Columbia University, New York 70027

Cultures of established and transformed fibroblasts were less able to contract a hydrated Collagen gel than normal precrisis cells. Postcrisis fibroblasts from different rodent strains and species underwent a further reduction in contraction ability after either spontaneous or simian virus 40 (SV40) transformation . Human precrisis fibroblasts contracted much more efficiently than two SV40-transformed human lines. Fibroblasts from a patient with Glanzmann's thrombasthenia were intermediate between all other human fibroblasts assayed and the SV40-transformed human lines. The absolute efficiency of contraction was dependent on temperature and serum concentration, but no conditions were found that resulted in equal efficiencies for the three types of cells. Precrisis cells were extremely sensitive to the passage procedures when assayed for Collagen contraction. ABSTRACT

The in vitro culture conditions routinely used to distinguish normal from transformed cells do not precisely reproduce an in vivo environment . Fibroblasts are in stromal collagen . Plastic or agar (13, 15), even with the addition of fibronectin (29), is a poor imitation of their in vivo situation . Although it is in no way identical to a tissue, a gel of native collagen more nearly duplicates the fibroblast's normal environment than does monolayer culture on a plastic dish. Indeed, Reid and Rojkind (20), Yang et al. (30), and others have reported that the culturing of cells on or in a collagen matrix helps to maintain their differentiated function. Fibroblasts in the dermis, dispersed in a collagenous matrix, are capable ofcontraction (7, 8). When wounding occurs, there is a localized proliferation of the fibroblasts followed by a contraction of the tissue to close the wound. Therefore, the ability of fibroblasts to contract collagen fibers may be characteristic of their normal interaction with the environment . Disturbance of this interaction could result in the abnormal tissue repair found in Glanzmann's thrombasthenia (21). Several years ago, Niewiarowski et al. (14) found that fibroblasts could cause the contraction of a fibrin clot in vitro . Dolfme et al. (4) reported that the spontaneous transformation in vitro of human fibroblasts resulted in a partial loss ofthis retractability. More recently, it has been reported by Bell et al. (1) that human fibroblasts are capable of contracting a hydrated collagen gel. We have assayed the ability of a large number of different cells to contract collagen, as an indication of their capacity to interact°with their normal environment. We find that establishment leads to diminished capacity and that tumorigenic lines are even more deficient in this capacity. 304

MATERIALS AND METHODS Cells and Culture A large number ofcells from various sources were used . Established cells have been previously described (17, 25, 26). Human precrisis cells were received from Dr. M. Lipkin at Sloan-Kettering Cancer Institute, New York and Dr . E. Pearlstein, New York University Medical Center, New York . SV80 and NG8 are SV40-transformed human cell lines obtained from Dr . S. Shin, Albert Einstein College of Medicine, New York . The SV40 dl 884transformants 8842A, 3A, and 7A were received from Dr . W. Topp, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. Lines AD and AA were received from Dr. S. Chen (Columbia University) . All human and rat cells had been grown on plastic dishes in Dulbecco's modified Eagle's medium (DME) (Grand Island Biological Co ., Grand Island, N. Y. [GIBCO] H21) and 10% fetal calf serum (Reheis Co ., Inc., Phoenix, Ariz .) before assay. Mouse cells had been grown in DME and 10% calf serum (GIBCO). These same sera were used for collagen gels . Culture conditions and procedures for passaging cells have been described (26). "3T3" and "3T6" passage protocols were essentially as described (27) for precrisis mouse embryo fibroblasts .

Collagen Partially purified collagen solution was prepared from rat tail tendon bundles according to the procedure of Bell et al . (1). Determination of protein concentration was by the method of Lowry et al. (12), using purified collagen (Collagen Corp .) as a standard. Final collagen concentration in all gels was 15014g/ml .

Preparation of Gels Medium and collagen were mixed with a counted number of cells in a small tube and 0.5 ml of the mixture was immediately plated in duplicate in 16-mm tissue culture wells (Linbro Scientific, Hamden, Conn.) that had been precoated with a film of 0.66% agarose to prevent cell attachment to the plastic. Each tube contained 0.4 ml of cells in DME plus 10% serum, 0.26 ml of 2 x DME plus 20% THE JOURNAL OF CELL BIOLOGY " VOLUME 87 OCTOBER 1980 304-308 © The Rockefeller University Press " 0021-9525/80/10/0304/05 $1 .00

serum, 0.09 ml of 0.1 N NaOH, and 0.3 ml (500 jug/ml) of collagen solution. After plating, thegels were incubated at 37°C in a 90°ío air, l0% CO z atmosphere. Solutions containing collagen formed a gel within 10 min of plating.

Measurement of Gel Size Gel contraction did not occur in the absence of cells. Most contraction occurred within 24 h when high concentrations of cells were used . Diameters of contracted gels were routinely measured at 24 h after plating to minimize any difference in cell number that might result from differences in growth rates in collagen of the various cell types. Measurements were done on a Wild M3 dissecting microscope, usingdarkfield illumination . This procedure makes it very easy to visualize theedge of apartially contracted gel. Contraction occurred more rapidly as the cell number was increased. Contraction ability was thus defined as the number of cells required to reduce the gel to one-half its original surface area . 24 h after plating (NA,/2)


Collagen Contraction by Different Cell Types

Fig. 1 shows the appearance of a completely contracted collagen gel in a 16-mm well . Uncontracted gels fill the wells in the form of a thin disk. The gel floats in liquid medium released from the gel during contraction . Faint vertical lines at the top of the well are 1-mm delineations from a ruler placed under the well (see Materials and Methods) . Fig. 2 shows the amount of contraction of a collagen gel as a function of the number of cells present per gel. Three different types of cells were used : normal precrisis mouse embryo fibroblasts (MEF), normal established mouse cells (3T3), and SV40transformed mouse cells (SV 101). The solid lines reflect the mean values from at least five separate experiments . Noncontracted gels had a surface area of 200 mm' compared with 5 mm' for fully contracted gels . Bars indicate the SD in number of cells for each cell type at the value NAt/2, where the gel is 100 mm2 . All three cell types were capable of causing contraction, but

Appearance of fully contracted collagen gel. 3T3 was plated at 10" cells/well . The photograph was taken 24 h after plating with low power on a Wild M3 dissecting microscope and dark-field illumination . Final magnification, X 3 . Bar, 3 mm . FIGURE 1


Relationship between contraction of collagen gels and the number of precrisis, established, and transformed fibroblasts plated in the gels . Gel diameters were measured 24 h after plating (see Materials and Methods) and values were converted to areas. Uncontracted gels have a surface area of 200 mm 2 . The NA,/2 is the number of cells required to contract the gel to 100 mm 2, one-half the original area. Bars indicate the SD for each cell type at NA,/2 . ", precrisis mouse embryo fibroblasts (MEF Swiss) ; X, established mouse fibroblasts (3T3 Swiss) ; O, SV40-transformed fibroblasts (SV101 Swiss) .

the number of cells required was very different . The transformed cells, SV 101, were extremely inefficient when compared with the precrisis MEF . The number of cells necessary for contraction to half the area in 1 d (NA, /2 ) for MEF was 5 X 103 , whereas for SV 101 it was 1 .8 X 10 5 . 3T3 was intermediate, with an NA t/2 of 3 X 104. There was no overlap in the range of values for the three types of cells. The transitions from precrisis to established to transformed cell apparently result in sequential reductions in the ability to contract collagen gels . The NAt/2 for a large number of other rodent cells was determined to ascertain the generality of these differences . These results are shown in Table I . All of the precrisis cells had NAI/2 values of 3-10 X 10 3 , with a mean of 6.0 X 103. Thus, the NAI/2 of the Swiss MEF (Fig . 2) was characteristic of precrisis rodent cells . The mean NAI/2 for established cells was 58 X 103, 10-fold higher than that of the precrisis cells. Therefore, as a group, established cells are clearly deficient, as compared with precrisis cells, in their ability to contract a collagen gel . Spontaneously transformed cells had a mean NAI/2 of 150 X 10 3 , whereas the SV40-transformed cells had a mean NAI/2 of 160 X 103 . These two groups of cells, with the same ability to contract a collagen gel, are significantly less efficient than established normal cells, extending our initial observations with MEF, 3T3, and SV 101 (Fig . 2) . Two other groups of cells are shown in Table I . The small t of SV40 is eliminated by deleting the region 0.54-0.59 map units. Such deletions do not effect the size of the SV40 large T antigen (23). Four different lines established by the transformation of REF with the SV40 0 .54-0 .59 deletion mutant dl 884 (23) had an NAI/2 similar to that of normal established cells rather than wild type SV40 transformants. This is in keeping with other reports that dl 884 transformants ten! to RAPID COMMUNICATIONS


TABLE I Ability of Rodent Fibroblasts to Contract Collagen Gels Cells Precrisis


Spontaneously Transformed

SV40 wt transformed

SV40 d1884 transformed


Tissue or parent cell

NA,/ 2 x 10-3 *




Rat Mouse Mouse Mouse Mouse Mouse Mouse

Fisher Balb/c Swiss DBA NIH C58 AKR

Embryo Embryo Embryo Dermis Dermis Dermis Dermis

5 10

6.0 t 3.0

Rat-1 3Y1 3T3 3T3 3T3

Rat Rat Mouse Mouse Mouse

Fisher Fisher Balb/c NIH Swiss

Embryo Embryo Embryo Embryo Embryo

70 100 40 50 30

58 ± 28


Mouse Mouse Rat Rat Mouse

DBA AKR Fisher Swiss

DBA AKR Rat-1 FI'1-4 MEF

200 50 200 200 100


14B WTA SV101

Rat Rat Mouse

Fisher Fisher Swiss

Rat-1 REF 3T3

100 200 180

160 ± 53

884B 884.2A 8843A 884.7A

Rat Rat Rat Rat

Fisher Fisher Fisher Fisher


30 80 40 60

52 ± 22

FL' 1-4 FL' 3-8 FL SV

Rat Rat Mouse

Fisher Fisher Swiss

14B 14B SV101

Mean ± SD$

5 3

5 3 10

600 <1,000 200

600 ± 326

* Mean number of cells required to reduce gel surface area to one-half, as measured in Fig. 1 . $ Mean NA 2 and SD for each group of cells .

exhibit a phenotype of partial transformation (3, 24) . Certain T-antigen negative revertants of SV40-transformed rat cells have nearly normal growth control as assayed by saturation density, serum requirement, and anchorage independence (22, 26) . These cells did not, however, revert to a high efficiency of collagen contraction but rather had an NA 1/2 of 600 x 10 3 , similar to that of their fully transformed parent.

human cells had an NA r /2 significantly higher than that of any of the precrisis cells and comparable to that of SV40-transformed rodent cells .

Human Skin Fibroblasts

The clear difference in NA,/2 between precrisis and postcrisis normal rodent cells (Table I) revealed a change in the cell population's ability to interact with collagen sometime during the passage procedure leading to establishment. In mouse cells, crisis and establishment do not occur before passage 15 . Table III shows the effect of passage protocol and passage number on collagen contraction . Cells passaged by the "3T3" protocol (17) are never allowed to become confluent. Their NA T/2 was that of precrisis cells at passage 3 and 6, as expected. At passage 15, when these cells had become established, their NA T/ 2 was typical of an established cell line. When precrisis cells were passaged by a 3T12 (17) protocol, they were always confluent. Under these conditions, there was a significant increase in the NA, /2 by the third passage, although these cells did not become established until passage 19 . This change in NAT/2 was progressive with passage number . Apparently, even a short period of time in contact with other cells reduced the ability of precrisis cells to contract collagen . These data may explain some of the variability seen earlier

Two SV40-transformed human cell lines were compared with precrisis fibroblasts from either normal individuals or persons with various inherited diseases of cellular growth control (Table II) . Cultures 265A and 285 were from individuals with a parent carrying the autosomal dominant gene for adenopolyposis of the colon and rectum. The individuals were asymptomatic at the time of the skin biopsy and their cells were grouped with normal cells, but they have a 50% probability of developing the cancer. As a group, the normal precrisis cells varied more than did the rodent cells, as illustrated in Table I . There was no significant difference among cells from normal, asymptomatic, and symptomatic patients, with one exception . Cells from the patient with Glanzmann's thrombasthenia had an anomglously high NAT/2. This reduced efficiency may be of significance because Glanzmann's cells are unable to cause contraction of a fibrin clot (5), and persons with this disease exhibit abnormal tissue repair (21). SV40-transformed 306


Relationship Between Establishment and Contraction of Gel


Ability of Human Fibroblasts to Contract Collagen Gels



SV40 transformed

NA,/ 2



Disease state


1380 1381 189 301 167 265A

Lung Lung Skin Skin Skin Skin Skin

9 20 10 5 2 7 1



Normal Normal Normal Normal Normal Normal Adenopolyposis, asymptomatic Adenopolyposis, asymptomatic


Skin Skin

3 5

166 291

Skin Skin

Cystic fibrosis Lesch nyhan syndrome Adenopolyposis Adenopolyposis,









Glanzmann's thrombasthenia


x 10 -3 *


20 2

± SD$

7.0 t 6.1

7.5 ± 7.9



* Mean number of cells required for gel reduction, as determined in Fig. 1 . $ Mean NA,/2 value and SD for each group of cells . TABLE III

The Relationship Between Establishment of MEF and Collagen Gel Contraction Type of passage* 3T3-like


Passage number$ 3 6 15

3 9 15

NA, /2 x 10 "3 6 4 30§

Varying the concentration ofserum in the collagen gel also affected contraction (Table V). All three lines showed an increase in efficiency (lower NA t/2) when plated in 15% serum and a marked decrease in efficiency when the serum contraction was lowered . This response parallels the growth response of these cells. However, the extent of variation was different for the three types of cells . MEF showed a 10-fold range in NA1 /2, the smallest deviation seen, with a continuous increase in NA 1/2 as the serum concentration was lowered . The 3T3 cells showed the greatest range of NA 1/2 values, 100-fold. At a concentration of 15% serum they were able to contract the gel and MEF, but at 0.1% serum they were 12-fold less efficient than MEF . Even 15% serum was not sufficient for SV101 to contract the gel at, a level comparable to MEF but did reduce the NA 1/2 to that seen for normal established cells in 10% serum (see Table I). At 0.1% serum, the NA1/2 of SV101 was one-thirtieth that seen with 15% serum. At no concentration of serum was the difference between normal and transformed cells abolished. DISCUSSION We have found clear differences in the abilities ofvarious types of fibroblasts to interact with and contract a hydrated collagen gel. These distinctions are not a function of species . Rather, they appear to reflect primary differences among precrisis, established, and transformed cells. We generally consider established cell lines such as 3T3 and Rat-1 to be "normal" because by many criteria they are comparable to primary cells (22). However, by the criterion of collagen contraction they are clearly not normal, because their NA1/2 is intermediate between that of precrisis cells and that of fully transformed cells. Human fibroblasts also appeared to fall into three classes. In the first class, normal human fibroblasts and fibroblasts from several people with unrelated inherited diseases had a relatively low NA1/2. In the second class, the Glanzmann's thrombasthenia cells had an NA 1/2 intermediate between that of normal TABLE IV

Effect of Temperature on Collagen Gel Contraction


20§ 30§

* Number of passages before cells were used for assay. All cells were assayed at one time in the same experiment . $ Passaged every 3 d at 3 x 105 cells/60-mm dish for 3T3, passaged every 3 d at 12 x 105/60-mm dish for 3T12 . § Significantly different from precrisis cells, P < 0.001 . 11 Significantly different from early passage "3T3" P< 0.002 .

with precrisis cells (Tables I and II) because in small areas of the dishes cells became confluent during routine culture. Effects of Culture Conditions on Collagen Contraction We chose MEF (Swiss), 3T3 (Swiss), and SV101 (Swiss) cells as a set of related mouse cells to investigate the effects of changing culture conditions. The effects of temperature are shown in Table IV. With all three types of cells, reducing the temperature to 22°C decreased the efficiency of contraction . Raising the temperature to 39.5*C enhanced the contraction efficiency of the MEF and 3T3, but did not affect SV101. 3T3 cells were much more susceptible to increased temperature than to decreased temperature.

Relative contraction efficiency* Cells

22 °C


37 °C

39 .5 °C

MEF 3T3 SV101

0.12t0.4 0.50±0.05 0.12±0.04

0.45t0.2 1 .10±0.01 0.20±0.08

1 .0 1.0 1 .0

3.0±1 .0 12 .2±3 .1 1 .0±0 .01

* Calculated as NA,/2 37°C/NA,/2 experimental temperature. NA, /2 were determined from titration curves as seen in fig. 1. The higher the number, the more efficient the contraction. TABLE V

Effect of Serum Concentration on Gel Contraction NA, /2


10 -3 in calf serum*







MEF 3T3 SV101

30 380 1500

15 140 700

10 16 100

6 25 100

3.6 3.4 50

* Cells were titrated in collagen gels containing each concentration of serum to determine NA812, as shown in Fig. 1 . A low NA,/2 reflects a high efficiency of contraction. t Standard conditions in other experiments. RAPID COMMUNICATIONS


and that of transformed cells. This disease is marked by a poor ability to effect wound healing (21). Our results suggest the possibility that fibroblasts from such persons are defective in their interaction with collagen. In the third class, the two SV40transformed human lines were very inefficient at contracting collagen, just as the SV40 transformed rodent cells were . Whereas the intrinsic differences among the normal, established, and transformed cells resulting in the altered abilities to contract collagen must be determined, we consider the following to be reasonable contributing factors: (a) Cells differ in their distribution of actin. Transformed cells have fewer actin cables (18). Changes in actin patterns could alter the tension on the substrate created by fibroblasts (11) . There are also small but reproducible differences in the size and distribution of actin cables between precrisis and postcrisis cells (28) . (b) Extracellular protease produced by transformed cells (16) could reduce the affinity of the cells for their collagen substrate. It could also alter the actin cables and the morphology of the fibroblasts, as do exogenously added proteases (19, 31). However, preliminary mixing experiments with transformed and precrisis cells suggest that the NA I/2 are approximately additive . No trans-active inhibition of contraction was detected (data not shown) . (c) Alteration or loss of collagen-binding proteins on the surfaces of the cells could also affect contraction. Interaction between the cell surface and collagen or collagen-glycopeptide complexes has been previously reported (6, 9, 10). Because MEF, 3T3, and SVI01 cells responded in a similar though not identical manner to changes in culture conditions, we suggest that these culture conditions do not affect the inherent ability of a given class of cell to interact with and contract its collagen substrate. Changes in temperature or serum concentration modify this interaction but do not abolish the intrinsic differences among the three types of cells. Not only did cells demonstrate an altered NA I/2 after undergoing the transition to established and transformed states, but even a short period of time in culture when cell-cell contacts and crowding occurred affected their subsequent interaction with the collagen gel. This is especially significant when one considers the report by Boone et al . (2) that even a few passages on plastic enhance the potential tumorigenicity of cultured ffbroblasts . Apparently, the way in which precrisis cells are passaged may affect their normality, even after a few passages . We would like to thank Marisa Bolognese for her help in the preparation of this manuscript . This work was supported by grants CA 25096 and CA 25066 from the National Cancer Institute. Receivedfor publication 27 May 1980, and in revisedform 25 June 1980. REFERENCES 1 . Bell, E ., B . Ivarsson, and C . Merrill. 1979 . Production of a tissue-like structure by



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