Neoplastic Conversion of Preneoplastic Syrian Hamster ... - Europe PMC

Vol. 3. No. 5

MOLECULAR AND CELLULAR BIOLOGY, May 1983. p. 931-945 0270-7306/83/050931-1502.00/0 Copyright ©C 1983, American Society for Microbiology

Neoplastic Conversion of Preneoplastic Syrian Hamster Cells: Rate Estimation by Fluctuation Analysis BRIAN D. CRAWFORD,l*t J. CARL BARRETT, AND PAUL 0. P. TS'01 Division of Biophvsics, Thle Johns Hopkins Unis'ersity Sch0ool of Hygiene and Puiblic' Healthl, Baltimore, Marvland 21205,1 and Environmental Carcinogenesis Grouip, Laboratory of Plulmonary Fuinction and Toxicology, National Institlute of Environmental Health Sciences, Research Triangle Park, Northl Carolina

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Received 25 August 1982/Accepted 17 February 1983

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete ste,ps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10-8 to 10-7 variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed. Neoplastic development, as described originally by Foulds (27-29), involves the stepwise evolution of a tumor cell toward increasing autonomy in vivo by successive qualitative alterations in the cellular phenotype. The progression of cancer is thus characterized by the emergence of new variant cell subpopulations that have a selective advantage for growth in their hosts (23, 25, 27-29, 41, 44). The mechanism(s) by which these variant cells arise is not, however, well understood. The observation that many carcinogens are mutagenic has supported the theory that neoplastic transformation may be the result of somatic mutation (reviewed in 6, 9, 43; J. C. Barrett and E. Elmore in L. S. Andrews, R. J. Lorentzen, and W. G. Flamm, Handbook of Experimental Pharmacology, in press). The demonstrated correlation between hereditary predisposition to cancer and several genetic defects affecting the repair of DNA

damage has provided supportive evidence that gene mutation may play a significant role in the process of neoplastic transformation. Several studies from this laboratory have shown that damage directed specifically toward DNA is sufficient to initiate neoplastic transformation and to induce single-locus somatic mutation in cultures of diploid Syrian hamster embryo (SHE) cells (10, 53). A direct comparison, however, of the processes of in vitro neoplastic transformation and somatic mutation in this cell system indicates that neoplastic transformation of normal diploid SHE cells cannot be explained completely by a single-locus somatic mutation in either X-linked recessive or autosomal dominant or codominant gene loci (6-8; Barrett and Elmore, in press). Rather, neoplastic transformation of SHE fibroblasts is characterized by the progressive stepwise emergence of cellular subpopulations with phenotypic alterations frequently associated with neoplasia (3, 7). It has been possible to identify intermediate preneoplastic cells that exhibit some, but not all, of the

Present address: University of California Los Alamos National Laboratory Genetics Group, Los Alamos, NM 87545.

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phenotypes displayed by neoplastic cells. Thus, in contrast to commonly studied gene mutations in mammalian cells, which occur in a single step (wild type to mutant), neoplastic transformation of cells in vitro appears to be a multistep process (6-8; Barrett and Elmore, in press). Analaysis of the role of single-locus mutations in the multistep process of carcinogenesis requires that discrete steps in neoplastic development first be dissected; only then can the genetic basis for specific alterations in cellular growth control be determined. The detection and definition of preneoplastic cells arising during the transformation process is therefore important. We have used the SHE cell model of neoplastic progression in vitro for this purpose. Cultures of SHE cells routinely can be maintained for up to 20 passages (60 to 80 population doublings) before senescence. Early-passage cells generally are diploid, have low fibrinolytic activity, appear morphologically normal, demonstrate no measurable colony formation in soft agar, and are not tumorigenic (1, 3, 4). Spontaneous transformation of SHE cells is infrequent; however, approximately 1 out of 10 embryo cultures maintained in our laboratory has escaped senescence and become an established cell line (3). These lines typically develop after a crisis period, usually 1 to 2 weeks, which is characterized by a decreased growth rate and a nearly complete loss of cloning efficiency by cells within the population. Recovery is marked by the appearance of more vigorously growing clonogenic cells capable of indefinite growth in culture. Barrett et al. (3) demonstrated that these established cultures differ from early-passage SHE cells. They are aneuploid and generally exhibit increased cloning efficiency (10 to 20%), and the majority of colonies possess elevated fibrinolytic activity when examined by the fibrin-agarose overlay technique. When such lysis-positive colonies (designated fibrin-overlay lysis positive, or FOL+ cells) were isolated, the clonal lines derived were non-tumorigenic when injected as cell suspensions in newborn hamsters or athymic mice (1, 7). It was demonstrated, moreover, that when FOL+ clonal cell lines were isolated from cell cultures previously treated with chemical carcinogens, these cells underwent a further transformation to neoplastic cells, marked by anchorage-independent growth and tumorigenicity when injected in suspension in newborn hamsters (1, 3, 7). These results were interpreted as evidence for a multistep process for the transformation of SHE cells in culture. By this hypothesis, FOL+ cells represent a population of preneoplastic cells which have a propensity to become neoplastic under appropriate conditions (1, 6). These isolated clones of preneoplastic cells can be used to study the

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mechanism(s) of conversion from the preneoplastic to the neoplastic state and the role of gene mutation in this process. Characterization and quantification of the cellular change(s) accompanying the neoplastic conversion of cells in culture requires the identification of phenotypic alterations that are highly correlated with tumorigenicity in vivo. The ability of cells to grow progressively while suspended in semisolid medium has been shown to distinguish tumorigenic cells from their nontumorigenic counterparts in a variety of cell systems (4, 11, 15, 16, 23, 24, 30, 34, 35, 45, 46, 48, 49, 55), although exceptions to this correlation do exist (31, 33, 42, 50-52). This phenotypic marker is very useful for studies with SHE cells because anchorage-independent growth in semisolid agar under defined growth conditions (36, 40) is highly correlated with the tumorigenic potential of Syrian hamster fibroblasts (4). The present study of the preneoplastic SHE cell system further confirms that conversion to anchorage independence is highly correlated with, and perhaps essential for, acquiring tumorigenicity. In this report, we have assessed the random origin of anchorage-independent variants of a preneoplastic anchorage-dependent cell line and have measured the rate of appearance of these variants by fluctuation analyses. The results of these investigations are discussed in relation to the multistage nature of neoplastic development. MATERIALS AND METHODS Cells and growth medium. The cell culture medium used was IBR Dulbecco modified Eagle reinforced medium (Biolabs, Northbrook, Ill.) supplemented with NaHCO3 (0.22 g/100 ml) and 10% Rehatuin fetal bovine serum (Reheis Chemical Co., Kankakee, Ill.) without antimicrobial agents. Cells were transferred by gentle trypsinization with 0.1% trypsin solution (1:250; GIBCO Laboratories, Grand Island, N.Y.) for 5 min at 37°C. All cells were tested by Microbiological Associates (Bethesda, Md.) and found free of mycoplasma contamination. SHE cell cultures were established from 13-daygestation fetuses collected aseptically by cesarian section of inbred golden Syrian hamsters of strain LSH/ss LAK (Lakeview Hamster Colony, Newfield, N.J.). Pools of primary cultures from littermates were stored in liquid nitrogen. Secondary cultures were initiated from frozen stocks, and all experiments were performed with tertiary or later cultures. Mutation experiments. The condition for the selection, quantitation, and characterization of ouabainresistant and 6-thioguanine-resistant mutants of Syrian hamster cells have been described (2, 54). These methods were adopted for the selection of variant cells mutated at the Na+/K+ ATPase and the HPRT gene loci, respectively. Fibrin-agarose overlay. To detect colonies of cells exhibiting enhanced proteolytic activity, the fibrin-

VOL. 3, 1983

ESTIMATION OF NEOPLASTIC CONVERSION RATE

overlay method of Jones et al. was used, modified as described previously (3). Tissue culture dishes (100 mm; Falcon Plastics, Oxnard, Calif.) were plated with cells at the densities needed to obtain 100 to 200 colonies after 7 to 8 days of growth. Each culture to be overlaid was washed twice with calciumfree and magnesium-free phosphate-buffered saline to remove serum inhibitors of fibrinolysis. For each overlay, 2.0 ml of 2.5% (wt/vol) agarose (Miles Laboratories, Elkhart, Ind.) in water were mixed with 2.5 ml of twofold Eagle minimal essential medium (GIBCO) supplemented with the appropriate serum and freshly added 95% clottable fibrinogen (2 mg/ml). After the rapid addition of 25 ,ul of thrombin (500 U/ml in phosphate-buffered saline), the solution was mixed and poured over the plate. The agarose was allowed to solidify for 5 min at room temperature; the plates were then incubated at 37°C in a 5% CO-95% air atmosphere. Clear zones of lysis in the opaline fibrin layer were visualized with an immunodiffusion viewer (Scientific Products, Columbia. Md.). The number of clear lysis zones was expressed as a percentage of the total colonies on the plate, as determined by fixation with 100% methanol and staining with 10% aqueous Giemsa. Tumorigenicity studies. Cells were trypsinized and suspended in complete medium at various concentrations (10 to 107 cells per ml in logarithmic increments), and 0.1 ml was injected subcutaneously into nonimmunosuppressed neonatal littermates (1 to 3 days old) of outbred Syrian hamsters (Lakeview). At least four to six animals was used for each determination. Tumorigenicity studies of cells attached to polycarbonate boats (1 by 5 by 10 mm) were performed as described (1). All inoculated animals were observed weekly for 1 year for the appearance of palpable agarose

tumors.

Measurement of transformed phenotypes. (i) Measurement of growth rates and saturation densities. Cells were plated at a density of 105 cells per 60 mm of tissue culture in medium containing either 1 or 10% serum. The number of attached cells was determined 15 h after plating. Cell counts were determined with a Coulter counter (Coulter Electronics, Inc.. Hialeah, Fla.) after trypsinization of cell monolayers with 2 ml of 0.25% trypsin (1:250, GIBCO) containing 0.1% EDTA. Cell counts were performed daily for 10 days. during which time the medium was changed every 2 days. Logarithmic growth was observed for at least 4

days; the population doubling times were calculated from this portion of the growth curve. Saturation densities were measured 7 to 10 days after plating. (ii) Cloning efficiencies in liquid and semisolid media. To determine the efficiency of colony formation in liquid medium, 102 to 104 cells were plated in triplicate 100-mm tissue culture dishes containing 8.0 ml of medium supplemented with 10% serum. After 8 days of incubation at 37°C, the number of colonies containing over 50 cells was determined after fixation with 100% methanol and staining with 10% aqueous Giemsa stain. The efficiency of colony formation in semisolid medium was measured by the procedure described by MacPherson and Montagnier (38) as modified by Kakunaga and Kamahora (35). Cells suspended in 4.0 ml of 0.3% agar (Difco Laboratories, Detroit. Mich.)

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supplemented with complete medium and 0.1% BactoPeptone (Difco) were plated in 60-mm dishes over a layer of 0.6% agar containing complete medium. The Bacto-Peptone supplement was required for efficient colony formation by hamster cells when suspended in semisolid agar (36, 40). Plates were incubated at 37°C in 5% CO-95% humidified air for 14 to 28 days. Colony formation efficiency in semisolid agar (Aga' trait) was expressed as the percentage of total cells which formed colonies containing at least 50 cells. (iii) Quantitation of extracellular fibrinolytic activity. To measure the release of extracellular plasminogen activator, 5 x 105 cells were plated in 60-mm tissue culture dishes. After attachment, the cultures were washed twice with phosphate-buffered saline to remove serum inhibitors of fibrinolysis. Eagle minimal essential medium (2 ml) supplemented with 5% fetal calf serum treated previously with acid was then added, and the cultures were incubated at 37°C in 5% CO-95% air for 18 h. The resulting cell-free media obtained from the cultures were assayed for fibrinolytic activity by the release of soluble 3H-labeled fibrinopeptides from [3H]fibrin-coated plates (5). Released radioactivity was expressed as a percentage of the activity released by urokinase (Calbiochem-Behring, La Jolla. Calif.); 1 U of fibrinolytic activity was defined as that which released 10% of the radioactivity liberated by 100 U of urokinase in 2 h at 37°C. Each unit of fibrinolytic activity represents approximately 0.2 to 0.3% of the available counts present on the [3H]fibrin-coated plates.

RESULTS Derivation and characterization of spontaneously transformed preneoplastic SHE cell lines. To study the mechanism of clonal progression of preneoplastic cells to the neoplastic state, we isolated, from a spontaneously established SHE cell line, clonal strains (designated FOL+) derived as colonies exhibiting lysis in fibrin-agarose overlay plates. Examination of four clonal strains (FOL' 1, 2, 3, and 4) derived from one such established cell culture (at a population doubling level of approximately 90 to 100) revealed the following cellular alterations (Fig. 1 and Table 1): (i) increased cloning efficiency (14 to 19%); (ii) stably elevated fibrinolytic activity; and (iii) heteroploidy (modal range 74 to 85). The colonial morphology of these cells appeared normal (Fig. 2A), and monolayer cultures showed contact inhibition of growth at confluence (Fig. 2B), as evidenced by their stable uniform cell saturation density (7.0 x 104 cells cm-2) during the plateau phase in growth curve analyses. Initially after isolation, all four cell lines were non-tumorigenic when inoculated in newborn hamsters (Tables 1 and 2) and were incapable of colony formation in semisolid agar when 106 cells were tested. After additional growth in vitro, however, FOL+ clonal lines did exhibit anchorage-independent variants capable of growth in semisolid agar (Aga' trait, Fig. 2C). The frequency of anchorage-independent vari-

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CRAWFORD, BARRETT, AND TS'O

~~~~~~~~~Cloning

EMBM HAMSTER EMBRYO SYRIAN HAMlSTER SYRIAN CELLS (primary explants)

DiMoid, 2iN :44

Efficiency 10-20% n 1 CRISISy \

0-25 2bL