Defective organization of actin in cultured skin fibroblasts from patients ...

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May 2, 1977 - lignancy before theappearance ofa frank invasive or metastatic growth. Although many biochemical assays have been proposed as indicators ...

Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 3019-022, July 1977 Medical Sciences

Defective organization of actin in cultured skin fibroblasts from patients with inherited adenocarcinoma (adenomatosis of colon and rectum/cytoskeleton/cancer detection/immunofluorescence/inherited disease)

LEVY KOPELOVICH*, SUSAN CONLONt, AND ROBERT POLLACKt * Memorial Sloan-Kettering Cancer Center, New York, New York 10021; and t State University of New York at Stony Brook, Stony Brook, New York 11794

Communicated by S. S. Cohen, May 2,1977

ABSTRACT In the cytoplasm of well-spread cultured normal fibroblasts, actin is organized into a network of cables that run the length of the cell just inside the adherent cell membrane. A diffuse matrix replaces the cables in fibroblasts that have become tumorigenic as a result of oncogenic transformation. We have found a similar disruption in actin organization in cultured skin fibroblasts (passage 6-10) obtained by biopsy from patients with the inherited colonic cancer, adenomatosis of the colon and rectum (ACR). Because ACR is inherited as an autosomal dominant trait, about half the children of ACR patients will develop colon cancer, but they typically remain asymptomatic until at least the second- decade of life. Actin distribution within cultured cells from children of ACR patients was identical either to that seen in cultured cells from normal persons or to that seen in cultured cells from ACR patients. The two different patterns were independent of age, sex, drug treatment, or infections of the donors. Apparently, this class of colonic carcinoma is accompanied by a systemic aberration in the organization of fibroblast cytoplasm, and this aberration can be detected by immunofluorescent localization of actin within cultured skin fibroblasts, prior to manifestation of any colonic symptoms. At present it is rarely possible to detect persons at risk for malignancy before the appearance of a frank invasive or metastatic growth. Although many biochemical assays have been proposed as indicators of premalignant states or cryptic early tumors, most have eventually been shown to be related to age, sex, drug treatment, or other variables linked to, but not clearly indicative of, a malignancy (1). Two serious problems limiting attempts to detect preneoplastic states or a disposition to eventual neoplasia are the inherent low frequency of incidence of any single type of tumor and the difficulty of obtaining identical sample material from prospective or actual patients once they are located. Our approach to the first of these two problems has been to study in detail an inherited syndrome, adenomatosis of the colon and rectum (ACR) (2-5), whose ultimate manifestation by the third decade of life is colonic neoplasia. Because ACR is inherited as an autosomal dominant mutation, children of ACR patients represent a mixed population, about half of whom have the usual low probability of developing colonic neoplasms and the other half of whom have a probability of 1.0 (3). Thus, ACR families offer a chance to monitor children who have no oncological symptoms but who with certainty will develop them. Our approach to the second problem has been to concentrate on the in vitro properties of cells obtained by cutaneous biopsy from patients and their families (6-10) rather than on cells grown from biopsy material of tissues at risk ( 1-13) or of biopsied tissues per se. By using only cultured skin fibroblasts (SF) we are able to examine a single cell type under reproducible conditions for presumptive cellular differences between ACR+ and ACR- individuals. Abbreviations: ACR, adenomatosis of the colon and rectum; SF, in vitro cultured skin fibroblasts; FCS, fetal calf serum; EMEM, Eagle's minimal essential medium. 3019

Fibroblast Growth Control In Vitro. Fibroblast growth in vitro is regulated in response to at least three different environmental variables. In order for cell division to occur, the amount of serum present must be adequate, the cell density must not be too high, and the cells must be provided with a solid substrate on which to anchor and spread (14, 15). Agents such as tumor viruses or chemicals can cause the loss of sensitivity to one or all of these variables, to yield transformed cells (15). Some, but not all, transformations also confer a tumorigenic potential on the cells. For many transformants of rat and mouse origin, the ability to grow without an anchoring substrate (i.e., anchorage-transformation) is the event most closely correlated with tumorigenicity (16, 17). Many cell populations that lack serum-sensitivity and density-sensitivity but retain an anchorage requirement have been shown to be nontumorigenic (16). SF from ACR patients and from about one-half of their children have lost serum- and density-sensitive growth control in culture (6-9). Compared with SF from normal persons, ACR SF grow better in low serum concentration and grow to a higher density (6). However, the ACR SF are identical to normal SF in their failure to grow in the absence of anchorage and in their failure to form tumors in nude mice (6, 8). Infection of normal or ACR SF by Kirsten murine sarcoma virus yields fully transformed tumorigenic cell lines, and-this transformation is more efficient on ACR than on normal SF (10). Thus, the ACR mutation partially decreases the growth control of SF so that they resemble the stable partial transformants of murine fibroblasts generated by simian virus 40 (15, 18, 19). Actin Distribution in Cultured Cells. The distribution of microfilaments containing the major protein actin within the cytoplasm of a well-spread cultured fibroblast has been found by immunofluorescence with antibody to actin to be highly ordered (20-22). Actin is organized in bundles or cables up to 1 gm in diameter, running the length of a spread cell (22, 23). In many transformed murine and avian fibroblasts, actincontaining cables are decreased in size and number (21, 24, 25). Anchorage-transformed tumorigenic cells are found to lack all but the smallest cables (24, 25). In this report, we describe the distribution of actin cables within cultured skin fibroblasts from ACR patients and their families, as determined by immunofluorescence with antibody to actin. MATERIALS AND METHODS Source of Human Material and Skin Biopsies. SF were obtained from several patients and their relatives seen at the Memorial Sloan-Kettering Cancer Center. These donors in-

cluded (i) persons with clinically symptomatic ACR; (ii) the children of persons in group (i); and (iii) two classes of normal persons-(a) spouses of ACR patients and (b) healthy volunteers. No difference was ever detected between subjects in the two normal classes, and henceforth they will be referred to as normal subjects and their SF, as normal cells.

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Medical Sciences: Kopelovich et al.

Proc. Natl. Acad. Sci. USA 74 (1977)

t E-

FIG. 1. Actin distribution in human skin fibroblasts. Fibroblasts growing from cutaneous biopsies were cultured for 4-12 passages in vitro and then fixed and stained with rabbit antiactin and fluorescein isothiocyanate-conjugated goat antiserum to rabbit IgG to visualize the distribution of actin in the cytoplasm. By focusing on the edge of the cell, actin distribution at the adherent side of the cell was kept in focus. The majority of fibroblasts obtained from patients with ACR (a and b) and from normal controls (c and d) differed in actin distribution. The normal cell populations had more cells with cables (Table 2). (Bar equals 10 am.)

Processing of Skin Biopsies. Subepidermoid biopsy specimens were taken from normal-appearing flat skin as described (6). The specimens were minced into 7 to 10 fragments and transferred to a culture vessel that contained 4 ml of culture medium (see below) supplemented with 25% (vol/vol) fetal calf serum (FCS). The vessel was incubated for 3 weeks; medium was changed twice weekly. A large number of dermal fibroblasts grew out around the explant as a monolayer during this incubation period. Cells in the outgrowth were suspended with trypsin and replated at 2-3 weeks and again about a week later

when the primary subcultures had become confluent. Thereafter, SF were passed at confluence at a subculture ratio of 1:3. Experiments were carried out between the 5th and 12th pas-

sages. Culture Conditions. Experiments were carried out in Eagle's minimal essential medium in Earle's balanced salt solution supplemented with 2 mM glutamine, 1X nonessential amino acids, penicillin (100 units/ml), and streptomycin (100 Ag/ml) (EMEM); to this was added 15% FCS. Cultures were routinely checked for bacterial contamination and mycoplasma (26) and were negative. - Preparation of SF for Determination of Actin Distribution. Unless otherwise noted, SF grown to about 75% confluency in T-75 flasks were harvested by trypsinization (0.25% trypsin/

EDTA; Gibco, cat. no. 630) for 10 min. The cells were washed twice by suspension and low-speed centrifugation in EMEM/ 15% FCS and plated out on sterile cover slips (11 mm diameter) in wells (Linbro; 24-well Disposo-tray, cat. no. FB16'24 TC) in 1.0 ml of EMEM/15% FCS. The SF were seeded at an initial plating density of 0.4 X I 04 cells per cover slip (about 25% confluency) and were examined for evenness of distribution on the cover slip, homogeneity of morphology, and sterility. After a 48-hr incubation in EMEM/15% FCS, the medium was changed to EMEM/1% FCS for 48 hr and then the cells were fixed by exchanging the medium for 3.8% formaldehyde in Ca2+- and Mg2+-free phosphate-buffered saline, pH 7.0, at room temperature. Immunofluorescent Localization of Actin in SF. Cover slips were kept in formalin for 4-8 days at 40 and then acetoneportfixed, stained sequentially with rabbit antiactin (1:80 in the same phosphate-buffered saline) and fluoroscein isothiocyanate-conjugated goat antiserum to rabbit IgG (1:20 in phosphate-buffered saline), and mounted cell-side down in Aquamount (27). Stained cells were scanned with a Zeiss photomicroscope with epi-illumination and an X63 objective. Cells were scored as positive for actin cables if fluorescent bands were seen to run the length of the cell when the edge of the cell was in focus. More than 100 cells were scored on each cover slip, and

Medical Sciences:

Kopelovich et al.

Table 1. Distribution of actin cables in SF from ACR phenotype

Proc. Natl. Acad. Sci. USA 74 (1977)

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Table 2. Analysis of distribution of actin cables in SF from ACR phenotypes

%-docells wit' Individuals M.E. A.F. T.F. V.F. M.G. A.H. V.M. J.P. Y.P. H.S. J.S. D.B. P.F. W.G. B.M. J.H. S.H. W.H. B.B. V.E. R.M. R.M.

R.E. B.P. J.S. F.M. L.A. F.L. S.S. CRL1221 CRL1225 *

Age

Sex

Symptomatic ACR M 31.0 (3) 17 M 31.6 (3) 19 M 49.3 (10) 36 23.4 (10) F 31 F 12.5 (2) 58 12.5 (2) M 37 25.5 (2) M 32 M 40.6 (7) 28 F 30.3 (4) 43 F 22.6 (3) 51 M 8.5 (2) 23 Asymptomatic progeny: positive 37.6 (3) M 5 M 19.0 (2) 17 40.5 (2) F 25 M 35.0 (2) 10 M 26.3 (3) 43 38.0 (2) M 48 M 25.0 (3) 47 Asymptomatic progeny: negative M 80 (1) 9 M 78 (1) 14 66 (1) M 8 F 80.0 (2) 32 Normals: spouses 71.8 (7) M 39 77 (1) F 51 M 92.0 (2) 40 M 69 (1) 61 Normals: healthy volunteers M 81.0 (3) 28 F 71.3 (3) 32 F 82.5 (2) 43 40 38

% of cells with No. of indi- No. of actin cables viduals exps. (mean + SEM)

actin cables, mean (n*)

M

91 (1)

M

78.8 (5)

Number of experiments.

alrexperiments were scored in ignorance of the origin of the cells examined (27). RESULTS Immunofluorescent Visualization of Actin Microfilaments in Human SF. The following conditions were found to be essential to minimize nonspecific variation from culture to culture. (i) Before transfer to cover slips, cells were grown to about 75-80% confluency after a 1:3 split. (ii) Passage number for cover-slip culture was kept between 5-12 because human cell strains beyond this stage are likely to undergo crisis (26). (iii) Cell trypsinization prior to plating on cover slips was carried out for 10 min at 370 to provide total removal of cells from the culture dish; the trypsinized SF were then washed twice in EMEM/15% FCS to inhibit and remove traces of proteolytic activity. (iv) Plating density was kept at 0.4 X 104 cells per cover slip (11 mm in diameter), which was optimal for cell fixation. Thus, all SF were examined at sparce cell density and a nonlimiting concentration of serum. Under these conditions, the SF spread well and growth was not inhibited by in vitro regulation.

1. Symptomatic ACR 2. Symptomatic progeny ACR phenotypes Non-ACR phenotypes 3. Normals

P*

11

48

31.6 + 2.99

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