Increased Rate of Cell-Substratum Detachment of Fibroblasts from Patients with DuchenneMuscular DystrophyAuthor(s): Claudia KentSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 80, No. 10, [Part 1: Biological Sciences] (May 15, 1983), pp. 3086-3090Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/13719 .

Accessed: 02/05/2014 00:22

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact support@jstor.org.

.

National Academy of Sciences is collaborating with JSTOR to digitize, preserve and extend access toProceedings of the National Academy of Sciences of the United States of America.

http://www.jstor.org

This content downloaded from 62.122.72.190 on Fri, 2 May 2014 00:22:51 AMAll use subject to JSTOR Terms and Conditions

Proc. Natl. Acad. Sci. USA Vol. 80, pp. 3086-3090, May 1983 Medical Sciences

Increased rate of cell-substratum d4 patients with Duchenne muscular d

(human fibroblasts/trypsinization)

CLAUDIA KENT

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

Communicated by Edwin T. Mertz, February 17, 1983

ABSTRACT When skin fibroblasts from patients with Du- chenne muscular dystrophy were treated with trypsin in the pres- ence of divalent cations, they detached more rapidly from the sub- stratum than did fibroblasts from normal individuals of similar age, sex, and passage number. This difference was observed when either the time of incubation or trypsin concentration was varied. The ease of detachment of both normal and dystrophic fibroblasts varied somewhat with culture age and plating density, although detachment was always greater for fibroblasts from dystrophic individuals. If the trypsin treatment was carried out in the ab- sence of divalent cations, both types of fibroblasts detached rap- idly from the substratum, suggesting that a divalent-cation de- pendent cell-substratum adhesion mechanism is altered in Du- chenne muscular dystrophy fibroblasts.

Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, affects approximately 3 individuals in every 10,000 live male births (1). This X chromosome-linked, recessive disease is characterized by an early onset and pro- gressive muscle wasting, and death usually occurs before age 25. The nature of the defective gene product is unknown, but the high levels of muscle creatine kinase in the serum of pa- tients, even before symptoms of muscle wasting appear, sug- gests that the muscle plasma membrane is abnormally perme- able. A considerable number of studies on membranes from DMD muscle, erythrocytes, lymphocytes, and fibroblasts sup- port the theory that the DMD gene product results in defective membranes, although much of this evidence has been con- tested (2, 3).

The availability of cultured skin fibroblasts from DMD pa- tients should allow for more rapid progress in elucidating the defective gene product in DMD. The biochemical defects of a number of diseases, including familial hypercholesterolemia (4) and the various lysosomal storage diseases (5), were identified as a result of studies with skin fibroblasts, even though these diseases do not appear to affect connective tissue in the patient. There is, therefore, a reasonable possibility that some conse- quence of the defective gene in DMD is observable in fibro- blasts from these patients, even though DMD is not overtly a disease of connective tissue. I have noticed that, when mono- layers of fibroblasts are treated with trypsin under certain con- ditions, fibroblasts from patients with DMD detach from the substratum more rapidly than fibroblasts from normal individ- uals.

In this paper I report a method for quantitative measurement of this phenomenon and characterize some parameters that af- fect the rate of cell detachment.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. ?1734 solely to indicate this fact.

3080

:tachment of fibroblasts from ystrophy

MATERIALS AND METHODS

Materials. Powdered Ham F12 medium and Fungizone were obtained from Grand Island Biological (Grand Island, NY); fetal bovine serum was from KC Biological (Lenexa, KS); genta- mycin was from Schering; tissue culture flasks and dishes were by Corning or Lux; soybean trypsin inhibitor was from Calbio- chem-Behring; deoxyribonuclease I was from Sigma; tissue cul- ture trypsin was from Armour Pharmaceutical (Kankakee, IL); twice-crystallized trypsin was from Worthington-Millipore (Freehold, NJ). The specific activity of the crystalline trypsin was reported to be 191 units/mg protein, where 1 unit hydro- lyzes 1 pimol of p-toluenesulfonyl-L-arginine methyl ester per min at 37?C. The trypsin was not reassayed in this laboratory.

Cells. Strains of skin fibroblasts were obtained from the Hu- man Genetic Mutant Cell Repository, Institute for Medical Re- search (Camden, NJ) (code GM) or the Cell Repository of Neu- romuscular Diseases, Montreal Children's Hospital (Montreal, Quebec) (code MCH or WG). For simplicity the fibroblast strains are referred to as either N (normal) or D (DMD), followed by a number indicating the age of the donor individual. The re- pository codes corresponding to these numbers are: N < 1, MCH 18; N 5, MCH 48; N 6, MCH 50; N 6b, MCH 40; N 10, GM 3348; D < 1, WG 839; D 4, WG 358; D 5, WG 840; D 6, WG 448; and D 10, GM 3783.

Cell Culture. Cell stocks were grown in F12 medium sup- plemented with 15% fetal bovine serum without antibiotics in an atmosphere of 95% air/5% CO2. For experiments, cells were cultured in the same medium supplemented with gentamycin (10 jig/ml) and Fungizone (0.5 /ag/ml). Cell stocks were split 1:2 at weekly intervals by washing twice with 0.5 mM EDTA in Ca2+- and Mg2+-free Dulbecco's phosphate-buffered saline (buffer P), then incubating for 15 min at 37?C in 0.25% tissue culture trypsin in Ca2/Mg2 -free buffer P. The trypsin action was stopped by addition of culture medium. The cells were then centrifuged, resuspended in culture medium, and distributed appropriately. For experiments at defined plating densities, the cells were counted with a hemacytometer. Stocks of fibroblast strains were frozen by suspending cells from one-half to one 75- cm2 flask in 1.0 ml of dimethyl sulfoxide/culture medium, 1:9 (vol/vol), incubating 3-24 hr at -70?C, and then storing in liq- uid nitrogen.

Detachment Assay. The standard assay was as follows (vari- ations in the procedure are indicated in the figure legends): When cell stocks were confluent, they were treated with trypsin and seeded at 1.4 x 105 cells per 35-mm culture dish, 1.2 ml of medium per dish. Occasionally a fibroblast strain had not reached confluence a week after splitting; such strains were not used for the assay. Cultures were assayed 5 days after plating unless oth-

Abbreviations: DMD, Duchenne muscular dystrophy; buffer P, Dul- becco's phosphate-buffered saline.

This content downloaded from 62.122.72.190 on Fri, 2 May 2014 00:22:51 AMAll use subject to JSTOR Terms and Conditions

Medical Sciences: Kent

erwise indicated. Because no differences were found in plating efficiency or growth rate between normal and dystrophic cells, this protocol assured that the matched pairs of cells were always assayed at the same cell density and stage of growth.

Sets of cell cultures to be assayed were removed from the CO2 incubator immediately prior to assay, with no more than 15 dishes per set. (In later assays the cell cultures were fed 3- 4 hr before the assay with culture medium containing 20 mM Hepes (pH 7.4) to prevent the cultures from becoming too al- kaline after removal from the CO2 incubator.) At 1-min inter- vals between dishes, the medium was removed, the cells were washed twice with 1.0 ml of Ca2*/Mg2+-free buffer P, and 1.0 ml of complete buffer P containing crystalline trypsin (100 u/g/ ml) and deoxyribonuclease (10 ,ug/ml) (6) was added. The tryp- sin was stored as a frozen stock solution, 10 mg/ml in 1 mM HCI and was diluted into ice-cold buffer P just prior to the as- say. The deoxyribonuclease was routinely included to prevent aggregation caused by DNA released from any damaged cells, but its presence was not critical. The dishes were incubated for 15 min at 37?C in a shaking water bath set at 25 oscillations per min. The water level of the bath was about 7 mm above the platform and the dishes were weighted down with a No. 7 rub- ber stopper. The incubation was terminated by addition of 0.05 ml of soybean trypsin inhibitor (2 mg/ml in Ca2+/Mg2+-free buffer P); the dishes were then quickly rocked back and forth three times, and the supernatant fluid was removed and added to 0.1 ml of 0.1 M EDTA in Ca2+/Mg2+-free buffer P on ice. The cells remaining on the dish were scraped with a rubber policeman in 1.0 ml of Ca2+/Mg2+-free buffer P and then was added to 0.1 ml of 0.1 M EDTA in the same buffer on ice. The suspensions were kept on ice until cells were counted, at which time the cells were resuspended by trituration 10 times with a Pasteur pipet. These suspensions were diluted with 15 ml of Isoton II, capped, and inverted four times before the cells were counted twice with a Coulter Counter set at 1/1I = 1/A = 4, window width = 4 - 100.

Because cells that had been trypsinized in buffer P were somewhat aggregated, the data are reported as the percentage of particles detached rather than of cells detached. In some experiments the cell suspensions also were counted with a he- macytometer to determine the ratio of particle number to cell number. That ratio was not significantly different between the

l l 100

80

Be I / /

20 -0

0 20 20 r

Time of incu

FIG. 1. Effect of time of incubation with trypsin on cell-substratum det Unfed cultures of N 10 and D 10 cells, both passage 13. o, Normal fibrobla

Proc. Natl. Acad. Sci. USA 80 (1983) 3087

detached cells and those that remained on the dish for any one dish. Therefore, the percentage of particles detached is the same as the percent cells detached. (The extent of aggregation dif- fered, however, with different strains of fibroblasts.) When er- ror flags are shown in the figures, the ends of the flags rep- resent the values obtained from duplicate dishes. When no error flag is shown, only one dish was assayed. Where a line inter- sects the data point, the duplicate determinations gave the same value.

RESULTS

When cultured human fibroblasts from normal individuals were subjected to treatment with trypsin in the presence of Ca2+ and Mg2+, the rate of detachment of the cells from the dish was relatively slow. Fibroblasts from patients with DMD, in con- trast, detached from the substratum considerably faster (Fig. 1). Although the time course varied from one strain to another, the DMD fibroblasts always detached faster than did normal fibroblasts when sets of matched fibroblasts were cultured and assayed at the same time. Rapid detachment of DMD fibro- blasts occurred when the cells were treated with either crude tissue culture trypsin (not shown) or with pure trypsin. More- over, when the time of incubation was held constant but the concentration of trypsin was varied, the DMD fibroblasts de- tached at lower levels of trypsin than did normal fibroblasts (Fig. 2). When excess soybean trypsin inhibitor was added to the cul- tures before trypsin, fewer than 2% of either normal or DMD cells detached from the dish, indicating that detachment was caused by the catalytic activity of the trypsin.

The rate of detachment of both normal and DMD cells var- ied with the age of the cultures (Fig. 3). Again, the shape of the curves varied for different strains, but some general con- clusions could be made. First, cells in younger cultures tended to detach faster than in older cultures. Second, cultures that had been fed with a complete medium exchange two or more days before the assay usually detached faster than cells that had not been fed. Third, DMD fibroblasts always detached faster than did normal cells that had been treated in the same man- ner, although in some cases the difference was small, e.g., in the youngest cultures in Fig. 3. The rate of detachment of nor- mal and dystrophic cells also varied with plating density (Fig.

X I 1

50 10 20

bation, minutes

achment. (A) Unfed cultures of N 6 and D 5 cells, both passage 15. (B) 3ts; A, DMD fibroblasts.

This content downloaded from 62.122.72.190 on Fri, 2 May 2014 00:22:51 AMAll use subject to JSTOR Terms and Conditions

3088 Medical Sciences: Kent

100 -

A

80

60 -o 60

40

20

10 20 30 40

Trypsir

FIG. 2. Effect of trypsin .concentration on cell substratum detachment. incubation was carried out for 20 min. (B) Cultures (4 day) of N < 1 (passa assay with Hepes-buffered medium (pH 7.4). o, Normal fibroblasts; A, DM

4), with detachment being maximal in more densely plated cul- tures.

Divalent cations had a pronounced effect on the rates of de- tachment of normal and DMD fibroblasts (Table 1). The pres- ence or absence of divalent cations in the solution in which the cells were washed did not affect subsequent trypsinization in buffer P, but the divalent cations did affect detachment rates when they were included during trypsinization. In the absence of divalent cations and in the presence or absence of EDTA, both normal and DMD cells were completely removed from the dish in 15 min. If trypsinization was carried out in the pres- ence of Mg2+ or Ca2+ or both, the level of detachment in 15 min was dramatically reduced for normal cells but only slightly reduced for DMD cells. Divalent cations presumably afforded protection of a cell surface protein or of proteins from the ac- tion of trypsin in the normal cells but not in the DMD cells.

A summary of the detachment data obtained with several

100 -

80

60

-' 40 - ?,,

20- A

A, I r 60 80 100 120 140

Time after

FIG. 3. Effect of culture age and feeding schedule on cell-substratum de fibroblasts; A, A, and x, DMD fibroblasts; , unfed cultures; ---,cultur both passage 13. Symbols are as in A except that the cultures were fed onl

Proc. Natl. Acad. Sci. USA 80 (1983)

10 20 30 40

, units/ml

A) Unfed cultures of N 10 and D 10 cells, both passage 11. The trypsin ge 13) and D < 1 (passage 12) cells. The cultures were fed 3 hr prior to D fibroblasts.

fibroblast strains assayed under standard conditions is shown in Fig. 5. Some strains of fibroblasts are represented in Fig. 5A more than once; in Fig. 5B the values obtained for each strain of fibroblasts have been averaged. Statistical analysis of the data in either A or B shows that the difference between the mean values for the normal and DMD cells is significant, P < 0.001.

DISCUSSION

The experiments in this paper were begun after it was ob- served, during routine subculturing of these fibroblasts, that freshly trypsinized normal cells appeared to be more aggre- gated when compared to DMD cells. Indeed, increased ag- gregation of normal- fibroblasts has been quantified and re- ported by Jones and Witkowski (8). I had observed that the presence of EDTA in the solution in which the cells were washed prior to trypsinization appeared to prevent aggregation of both

B /

160 80 100 120 140

plating, hours

tachment. (A) N 10 and D 10 cells, both passage 12. o, , and a, Normal as fed at 72 hr; -.-, cultures fed at 72 and 142 hr. (B) N 6 and D 5 cells, y at 94 hr.

This content downloaded from 62.122.72.190 on Fri, 2 May 2014 00:22:51 AMAll use subject to JSTOR Terms and Conditions

Medical Sciences: Kent

100 - A B

80-

0 60

a. 40

20

40 80 120 160 40 80

Plating density, cells x 10-3/dish

FIG. 4. Effect of plating density on cell-substratum detachment. (A) Unfed cultures of N 6 and D 5 cells, passage 16. (B) Unfed cultures of N 10 and D 10 cells, passage 12. o, Normal fibroblasts; A, DMD fi- broblasts.

cell types, and some experiments were performed in which the divalent cation content of the washing and trypsinization so- lutions was varied. During those experiments it was observed that, if trypsinization were carried out in the presence of di- valent cations, the detachment of normal but not DMD cells was suppressed. I had experienced difficulty attempting to quantitate the cell aggregation, and this presumably was due to lack of a constant shear force as was used in the assay procedure of Jones and Witkowski (8). However, the detachment phe- nomenon could be quantitated easily and reproducibly with a Coulter Counter. Therefore, I proceeded to measure the ease of detachment of several strains of normal and DMD fibro- blasts and to characterize some parameters that affected the process.

The reason for the altered adhesive behavior of the DMD fibroblasts is unknown. It is possible that the extensive degen- eration occurring in DMD patients selects for a minor fibro- blastic cell type that exhibits altered adhesive properties in vi- tro. However, such a selection mechanism would be expected to result in more marked differences in cultures obtained from

Table 1. Effect of divalent cations on cell-substratum detachment

Prewash Trypsin % particles detached solution solution Normal Dystrophic EDTA Buffer P 23 71 CMF Buffer P 12 66 Mg Buffer P 25 76 Ca Buffer P 28 74 Buffer P Buffer P 30 83 CMF EDTA 98 98 CMF CMF 89 94 CMF Mg 36 82 CMF Ca 25 91 CMF Buffer P 21 83

Standard assay conditions were used except that the divalent cation content was varied as indicated. The fibroblast strains used were 4-day unfed cultures of N 10 and D 10 cells, both passage 14. The EDTA so- lution was 0.5 mM EDTA in Ca2+/Mg2+-free buffer P, here called CMF. "Mg" indicates 0.5 mM MgCl2 in Ca2 -free buffer P and Ca indicates 0.9 mM CaCl2 in Mg2+-free buffer P which are the concentrations of these cations in complete buffer P. The values shown are the averages of duplicate determinations.

Proc. Natl. Acad. Sci. USA 80 (1983) 3089

100

A A A^^ B ^4

X 80^ ^ Q),-A 10

60 ^ ^ U) A

^

40 oOO 06 n_ 4 0 - 0 0 06

000 010 0

20 o- 05 06 O0

to 0<(1

N D N D

FIG. 5. Summary of cell-substratum detachment data. (A) All as- says that were performed with all strains under standard conditions are shown. The age of the cultures varied from 3 to 6 days, and the passage numbers were from 11 to 16. * and A, Assays performed with cultures fed with Hepes-buffered medium (pH 7.4) 3 or 4 hr before the assay; o and A, assays performed with unfed cultures. (B) All assays for each strain have been averaged. The number of observations with each strain varied from 2 to 16. The ages of the donors for the fibroblast strains are indicated next to the symbols. The statistical test performed was the two-tailed unpaired Student t test (7).

older donors than from younger ones. In fact, DMD fibroblasts from a 10-year-old donor (D10) were not significantly more or less adhesive than fibroblasts from a 2-month-old infant (D < 1) who had not shown any clinical manifestations of the disease at the time of biopsy. Therefore, the cell selection mechanism is less likely than a mechanism involving altered cell surface properties that are more closely related to the defective DMD gene product.

At present, the relationship of decreased cell aggregation to the increased cell-substratum detachment behavior is un- known. In both processes DMD fibroblasts appear to be less adhesive than normal fibroblasts, but the molecular mecha- nisms for the two processes are not necessarily identical. In fact, the preliminary results mentioned above suggest that the two phenomena are distinct, since prewashing with EDTA de- creased cell aggregation (unpublished data) but had no effect on detachment (Table 1). The decreased cell aggregation and increased cell detachment are likely to be two different sec- ondary consequences of the defective DMD gene product.

The ease of detachment of both normal and DMD cells is undoubtedly a complex process that depends on several cell surface-associated molecules, which vary in concentration as a function of such parameters as culture age, plating density, and replacement of culture media. There are probably other com- monly variable environmental factors that will be found to af- fect this process. For instance, DMD cells cultured on glass coverslips do not detach much more rapidly than normal cells do, indicating that ease of detachment can be affected by the nature of the substratum. It should be possible to measure the concentration of certain cell surface molecules as a function of culture age, plating density, trypsinization conditions, etc., and to correlate changes in the amounts of these molecules with changes in detachment. Thus, it should be possible to deter- mine which cell surface molecule of the DMD fibroblasts is responsible for the abnormal detachment behavior.

The author thanks Sally Berry for excellent technical assistance. This research was supported in part by a grant from the Muscular Dystrophy Association of America. This is journal paper No. 9205 from the Purdue Agricultural Experiment Station.

This content downloaded from 62.122.72.190 on Fri, 2 May 2014 00:22:51 AMAll use subject to JSTOR Terms and Conditions

3090 Medical Sciences: Kent

1. Emery, A. E. H. (1977) in Pathogenesis of Human Muscular Dys- trophies, ed. Rowland, L. P. (Excerpta Medica, Amsterdam), pp. 42-52.

2. Rowland, L. P. (1980) Muscle Nerve 3, 3-20. 3. Tsung, P. K. & Palek, J. (1980) Muscle Nerve 3, 55-69. 4. Goldstein, J. L. & Brown, M. S. (1977) Annu. Rev. Biochem. 46,

897-930.

Proc. Natl. Acad. Sci. USA 80 (1983)

5. Neufeld, E. F., Lim, T. W. & Shapiro, L. J. (1975) Annu. Rev. Biochem. 44, 357-376.

6. Dulbecco, R. & Vogt, M. (1954)J. Exp. Med. 99, 167-182. 7. Remington, R. D. & Schork, M. A. (1970) Statistics with Appli-

cations to the Biological and Health Sciences (Prentice-Hall, En- glewood Cliffs, NJ), pp. 210-213.

8. Jones, G. E. & Witkowski, J. A. (1979)J. Neurol. Sci. 43, 465-470.

This content downloaded from 62.122.72.190 on Fri, 2 May 2014 00:22:51 AMAll use subject to JSTOR Terms and Conditions