IN VITRO Volume 14, No. 3, 1978 All rights reserved @
S T U D I E S ON T H E T R A N S F O R M A T I O N O F R A T E M B R Y O C E L L S O F L O W P A S S A G E BY C A R C I N O G E N I C F L U O R E N Y L H Y D R O X A M I C ACIDS AND T H E I R A C E T A T E E S T E R S H. KURZEPA, H. R. GUTMANN, D. MALEJKA-GIGANTI, A. KRAUSS, J. CERVENKA, G. J. VOSIKA, ANDR. E. RYDELL Laboratory for CancerResearch, VeteransAdministration Hospital; and the Departmentsof Biochemistry, OralPathology and Human and OralGenetics, Medicine, and Laboratory Medicine and Pathology, Universityof Minnesota, Minneapolis, Minnesota 55417
SUMMARY
Rat embryo cells of low passage subjected to a single treatment with certain carcinogenic fluorenylhydroxamic acids and their respective acetate esters showed signs of transformation in vitro, such as changes in phenotype, growth in soft agar and agglutination with concanavalin A. In addition, certain changes in karyotype and loss of diploidy were observed. There was no evidence, either by electron microscopy or by assay of RNA-dependent DNA polymerase, for the presence of virus. None of these cell lines produced tumors after inoculation into the isologous host. The results of this study lead us to suggest that malignant transformation is a multistep process and that certain criteria of transformation of rat embryo cells are associated with the initial stage(s) in which the cells are transformed without being tumorigenic. The ultimate test for malignant transformation of rat embryo cells remains the production of tumors in a susceptible host after inoculation of treated cells. Key words: transformation; rat embryo cells; fluorenylhydroxamic acids. INTRODUCTION
Investigations have been carried out in this laboratory for some time to establish a method for the screening in vitro of fluorenylamides and their activated derivatives by means of rat embryo cells. Assays in vitro have been applied to the studies of the effect of carcinogenic aromatic polycyclic hydrocarbons on hamster or mouse cell cultures [for review see Mishra and DiMayorca (1) and Heidelberger (2)]. Few reports have dealt with transformation of rat embryo cells in vitro by carcinogens in general (3-7) and specifically with transformation by carcinogenic arylamides or arylhydroxamic acids {6,8). A previous study from this laboratory has indicated that monolayers of rat embryo fibroblasts exposed to certain fluorenylhydroxamic acids and their acetates produced a high incidence of transplantable sarcomas when the cells were injected into isologous and nonisologous hosts (8k This investigation did not include quantitation of transformation. Moreover, the cells were from a commercial source and had undergone numerous passages (approximately 40) prior to multiple expos-
ures to the carcinogens. The present work was undertaken to determine whether induction of malignant transformation could be accomplished when low-passage rat embryo cells were treated with a single close of N-hydroxy-2-fluorenylacctamide (N-hydroxy-2-FAAk N-hydroxy-2-fluorenylhenzamide (N-hydroxy-2-FBA), N-hydroxy3-FAA, N-acetoxy-2-FAA, N-acetoxy-2-FBA and N-acetoxy-3-FAA. All of these compounds are potent carcinogens for the rat in vivo (9-11). Transformation in vitro was monitored by standard criteria (morphologic changes, growth in soft agar, agglutination by concanavalin A, karyotype analysis). The ultimate test for malignant transformation was the injection, into isolognus hosts, of cells derived from colonies that were judged transformed by the above criteria. MATERIALS AND METHODS
Media. Eagle's medium (MEM) containing Hanks' basal salt solution (BSS) was purchased from Microbiological Associates, Bethesda, Maryland. This medium was fortified with Lglutamine (2 mMk sodium pyruvate {1 mM), a
261
262
KURZEPA ET AL.
mixture of nonessential amino acids (0.1 mM) (Microbiological Associates, Bethesda, Md.)(12) and 10% fetal bovine serum (Grand Island Biological Co., Grand Island, N.Y.) inactivated by heating at 56 ~ C for 30 rain, and is designated fortified M E M . Cultivation of cells. Cells were obtained from 18-day-old embryos of a single Wistar-Furth rat purchased from ARS Spragne-Dawley, Madison, Wisconsin. The embryos were removed under sterile conditions, washed several times with Ca 2§ and M~§ BSS supplemented with penicillin (250 U per ml) and streptomycin (250/~g per ml) and minced. The mince was subjected to digestion by a solution of 0.25% trypsin. The digestion was carried out at 37 ~ C and repeated four times. The supernatant liquid from the first digestion was discarded. The subsequent supernataut liquids were combined and centrifuged. The resulting pellet was suspended in fortified M E M and the suspension was filtered through three layers of sterile gauze. An aliquot of the filtrate (4 ml) was delivered into 25-cm 2 flasks (Falcon Plastics, Los Angeles, Calif.) for cell propagation. The remainder was frozen in fortified M E M containing 15% glycerol and kept at - 8 5 ~ C for further use. The cells were incubated at 37 ~ C in 5% CO~:95% air (90% humidity). After 24-hr incubation of the suspension, the primary cells were rinsed with fortified M E M and replenished with the same medium. These primary cells were the source for all subsequent experiments. Compounds. N-hydroxy-2-FAA, m.p. 150151 ~ C (9); N-hydroxy-3-FAA, m.p. 131-132 ~ C (13); N-hydroxy-2-FBA, m.p. 189-191 ~ C (10); N-acetoxy-2-FAA, m.p. 111-112 ~ C (14); Nacetoxy-3-FAA, m.p. 104-105 ~ C (15); and Nacetoxy-2-FBA, m.p. 130-132 ~ C (11) were prepared by the published procedures. The compounds were dissolved in acetone (spectral grade) at a concentration of 0.5 or 1.0/~mol per ml. The acetone solutions of the hydroxamic acids or of the acetate esters were diluted 100 times with M E M or with BSS, respectively. Prior to use in the carcinogenicity tests, the stability of all compounds in M E M and BSS was tested by analysis of the medium with the use of high-pressure liquid chromatography (16,17). The recovery data indicated reasonable stability of the fluorenylhydroxamic acids in M E M during 24 hr. However, the acetate esters were not recovered from M E M indicating instability or rapid interaction with that medium. The recovery data showed greater stability of these compounds in BSS. Thus N-
acetoxy-2-FBA was recovered nearly quantitatively within 24 hr. Similarly major amounts of unchanged N-acetoxy-3-FAA were recovered within that period. However, only 10% of Nacetoxy-2-FAA was recoverable from BSS after incubation for 0.5 hr. The times of exposure of the cells to the compounds were selected according to their apparent stability. Exposure to compounds. Cells (104) of passages 5 to 7 were seeded on a plastic Petri dish with grid (60-mm diameter) and incubated for 24 hr in fortified MEM. The medium was removed and the cells were washed three times with BSS. The cells then were incubated with solutions of fluorenylhydroxamic acids and their acetates for 24 hr and 0.5 to 5 hr, respectively. Controls consisted of cells treated with M E M and M E M containing 1% acetone as well as BSS and BSS containing 1% acetone. Fifteen dishes were used for each concentration of compound and for the controls. After treatment with fluorenylhydroxamic acids, the cells were washed three times with MEM. After exposure to acetates, the cells were washed three times with BSS.
Determination of cytotoxicity and transformation. Cytotoxicity was estimated by cell count of several dishes immediately after exposure to the compounds. Cytotoxicity also was determined by plating efficiency (18). Transformation was assayed by the transformation frequency based on the number of morphologically altered colonies relative to the number of total colonies (18, 19). Morphologically altered colonies were isolated (20) and propagated in fortified M E M for further tests of transformation, such as growth in soft agar (21) and agglutination by concanavalin A (Con A) (22). For growth in soft agar, the cells from each altered colony and from the appropriate controls were grown on three to five dishes, and development of colonies was followed for 8 weeks. Agglutination was measured as described (23) except that the cells for the assay were washed with Ca 2§ and Mg2+-frec BSS and dispersed in 0.67 M phosphate buffer, pH 7.5 (10~ cells per ml). The extent of agglutination was calculated from the following expression: % A,-F agglutination ---- - - x 100, where A~ ---T,-F agglutinated cells in sample; T8 = total cells in sample; Ac = agglutinated cells in control; Tc = total cells in control; F = __At x Ts. Controls for T~ agglutination of untreated and carcinogen-treated
263
TRANSFORMATION OF RAT EMBRYO CELLS cells consisted of a cell suspension in p h o s p h a t e buffer without addition of C o n A. Test for malignancy of transformed cells. Confluent cultures derived f r o m t r a n s f o r m e d colonies a n d from the a p p r o p r i a t e controls were harvested by trypsinization with a 0.05% trypsin-0.05% E D T A solution (Microbiological Associates, Bethesda, M d . ) . After centrifugation, the ceils were w a s h e d with M E M , centrifuged a n d r e s u s p e n d e d in M E M . Approximately 107 cells per 0.1 ml M E M were injected subcutaneously into n e w b o r n W i s t a r - F u r t h rats or i n t r a m u s c u l a r l y into wean-
ling rats of the s a m e strain. T h e developed t u m o r s were excised a n d e x a m i n e d histologically. R a t s were m a i n t a i n e d on P u r i n a C h o w a n d water a d libitum for 8 m o n t h s after injection of the cells. Chromosomal analysis. N e a r - c o n f l u e u t cultures were trypsinized after exposure to colchicine for 2 to 4 hrs. T h e cells t h e n were treated with hypotonic KCI a n d subjected to fixation with acetic acid a n d methyl alcohol {1:1}. T w o sets of slides were prepared. One of these was stained with the conventional G i e m s a stain. T h e second set was treated with trypsin for G - b a n d i n g (24).
TABLE 1 TRANSFORMATIONOFR ATEMBRYOC ELLS{5THPASSAGE}AFTERAS INGLEEXPOSURETO F LUORENYLHYDROXAMIC ACIDS DETERMINED BYASSAYSIN VITROANDIN VIVO
Compound
Dose
Plating TransformaEfficiency a fion a
(nmol/ dish) (%) MEM
0
--d
Designation of Isolated Colony
Growth in AgghtinaSemisoft tion byc Agarb Con A
No. Passage Prior to Injection
Tumor Incidence
19 17 35 16
0/4 0/5 5/6 f 0/5
(%) 0e
-
-
N/7/6
-
-
Na/7/3 Na/7/4 Na/7/5
+ -
-
20 21 18 25 36
0/3 3/3 g 0/5 0/6 0/6
M E M + I % acetone
0
_d
N-Hydroxy-2-FAA
50
0.2
96-100
D'/6/1 D'/6/2 D'/6/3
4-44-4-+
++ +
16 16 24
0/6 0/5 0/12
25
0.5
90-100
D/7/4 D/7/8
+ +4-
+ -4-
15 18
0/4 0/5
25
2.0
100
C'/7/2
+'4-+
-4-
C'/7/4
-4-+
-4-
15 18 17 24 37
0/4 0/5 0/5 0/6 0/6
N-Hydroxy-2-FBA
N-hydroxy-3-FAA
0e
N/7/1 N/7/3
12
2.0
91-100
C/7/4
4-
-4-
17
1/13 h
50
1.0
97-100
A'/7/1 A'/7/6
+ +
+ 4-
22 17 26
0/5 0/5 1/6 i
25
2.0
97-100
A/7/2
4-
4-
16 34 A/7/8 4-4-4421 a Plating efficiency and transformation frequency was determined 22 days after the cells were seeded. b Symbols represent the average number of colonies on four or five Petri dishes: - : - 0-10; 4- = 10-50; 50-100; + + + = >100. c + Indicates 50% agglutination of cells. d Confluent monolayer. e No morphologic changes. f Five rats had a pleomorphic sarcoma at the site of inoculation. s Two rats had a Iibrosarcoma and one rat had a pleomorphic sarcoma at the site of inoculation. h One rat had a Wilm's tumor of kidney ~nephroblastoma). i One rat had a mammary adenocarcinoma.
0/5 0/5 0/5 ++=
264
KURZEPAETAL.
The modal number and ploidy for each cell line was established. Sets of karyotypes were prepared for detailed characterization of the chromosomal constitution of each cell line. Screening for presence of virus. Cells from untreated and carcinogen-treated cultures were examined routinely by electron microscopy for the presence of viral particles. In addition, a high speed (I00,000 x g) pellet of supernatant fluids from cultures that were subsequently inoculated into test animals was prepared and assayed in triplicate for RNA-directed DNA polymerase activity (reverse transcriptase) using the synthetic template Poly (rc) (dG)12.18 as previously described (25L
The results of transformation assays in vitro and in vivo are summarized in Table 1 and 2. No transformation was seen in the controls. An example of a control colony and a monolayer derived from this colony are shown in Fig. 1A,B, respectively. Transformation frequency by morphologic criteria (19,26,27) was almost quantitative after exposure to fluorenylhydroxamic acids. A typical picture of a transformed colony and a monolayer derived from this colony is given in Fig. 1C,D, respectively. Acetate esters produced morphologic transformation ranging from 2% to 29%, whereas no morphologic changes were noted in the controis (Table 2L There are reports in the literature that transformation of mouse embryo cells exposed to polycyclic aromatic hydrocarbons can be RESULTS graded into three classes based on the appearance Cytotoxicity as determined by cell count indi- of transformed loci. Only one of these classes is cated a 29% to 56% decrease in the number of tumorigenic (28). However, rat embryo cells that cells treated with fluorenylhydroxamic acids. The had been exposed to fluorenylhydroxamic acids corresponding decrease for cells exposed to aceand their acetates did not show morphologic altate esters was 37% to 66%. No decrease in the terations that would lend themselves to the gradanumber of cells was noted in the controls. Cytotion applied to transformed mouse cells. toxicity measured by plating efficiency 2 to 3 Variable clonal growth in semisoft agar was observed with all cells derived from colonies that weeks after exposure to the compounds ranged from 0.2 to 2% in cultures treated with fluorenyl- had been transformed morphologically by fluorhydroxamic acids (Table I} and from 1% to 5% enylhydroxamic acids and the corresponding esters (Tables 1, 2). A typical picture of a colony in cultures treated with the acetate esters (Table 2 }. Incubation of cells in M E M alone or in M E M in semisoft agar derived from cells exposed to Nhydroxy-2-FAA is seen in Fig. 2,4. In the majority containing 1% acetone showed no evidence of cytotoxicity (Table 1 ). On the other hand, incubaof cases, cells grown in control medium did not tion with BSS alone or with BSS containing 1% form colonies in soft agar. However, there were at least two colonies isolated from control medium acetone gave only plating efficiencies of 8% to which did grow in semisoft agar, although the 10% (Table 2L TABLE 2 TRANSFORMATION OF n AT EMBRYO CELLS ( 7TH P ASSAGEIAFTER A SINGLE EXPOSURE TO ACETATE ESTERS OF F LUORENYLHYDROXAMICACIDS DETERMINED BY ASSAYS IN VITRO AND IN VIVO
Compound
Dose
Plating TranslormaEfficiency a tiona
(nmol/dish) (%) BSS BSS + 1% acetone N-Acetoxy-2-FAA
Designation of Isolated Colony
No. Growthin Agglutina- Passage Semisoft tion by Prior to Agarb Con Ac Injection
Tumor Incidence
(%)
0 10 0d N/5/10/6 + 21 0 8 0d Na/0.5/10/2 _+ 22 50 4 2 E'/10/1 +++ + 25 25 5 2 E/10/1 -4-+ + 20 N-Acetoxy-2-FBA 25 1 6 F'/10/4 -4419 12 2 8 -f N-Acetoxy-3-FAA 50 1 29 B'/10/5 4-4424 25 3 7 _I a Plating efficiencyand transformation frequency was determined 13 days after the cells were seeded. b Symbols represent the average number of colonieson four or five dishes: - = 0-10; + = 10-50; + + = + + + = >100. c -4- Indicates 50% agglutination of cells. d No morphologic changes. e Two rats had a pleomorphic sarcoma at the site of inoculation. f These coloniesdid not survive subsequent propagation.
2/6 e 0/6 0/7 0/3 0/5 0/5
50-100;
TRANSFORMATION OF RAT EMBRYO CELLS
265
FIG. 1. Representative phase-contrast photographs of untreated and carcinogen-treated rat embryo cells. A, Colony of untreated cells (control). Note cells growing in a parallel-oriented pattern. B, Confluent monolayer derived from colony shown in A. C, Colony of cells treated with N-hydroxy-2-FAAas described in the text. Note loss of cellular orientation, crisscross pattern and irregular borders. D, Monolayer derived from colony shown in C. Note tendency to form multilayered loci. x150.
level of colony formation was low. Since similarly low levels of colony formation also were observed with morphologically altered colonies obtained by exposure to carcinogens, clonal growth in semisoft agar would not appear to be a reliable criterion for malignant transformation of rat cells in vitro. It seemed possible that morphologically transformed cells and bona fide tumor cells could be distinguished by quantitative differences in their growth in semisoft agar. Indeed, tumor cells derived from plcomorphic sarcomas induced in Wistar-Furth rats by subcutaneous injection of the carcinogen N-hydroxy-2-FBA exhibited generally a high colony formation in semisoft agar ranging from 500 to 11000 colonies per dish. A picture of a colony derived from tumor cells is shown in Fig. 2B. However, the variations in clonal growth between different batches of tumor cells were too large to establish a statistically significant number that would permit us to distinguish between tumorigenic and nontumorigenic cells. Moreover, there was an overlap between the lower range of the clonal growth of bona .fide
tumor cells and of the clonal growth of morphologically transformed cells that proved to be nontumorigenic by the inoculation test. As a third criterion of transformation, agglutination tests with Con A were carried out. However, no consistent pattern indicative of transformation was evident (Tables 1, 2). Thus, although the test appeared to be positive for cells that had been derived from morphologically altered colonies obtained after treatment of rat embryo cells with the carcinogens, there were also instances in which cells derived from control colonies gave 50% agglutination with Con A. Our previous data had shown that rat embryo cells that had undergone malignant transformation after multiple exposure to carcinogenic fluorenylhydroxamic acids or their corresponding acetates induced fibrosarcomas at the point of injection within 2 to 4 months after subcutaneous inoculation into isologous and nonisologous hosts (8). Accordingly, the rats injected with rat embryo cell lines in this study were observed for tumor development for as long as 8 months. Inoculation of
266
KURZEPA ET AL.
FIG. 2. Clonal growth in semisoft agar. A, Colony derived from cells exposed to the carcinogen Nhydroxy-2-FAA, x200. B, Colony derived from tumor cells. >
S T U D I E S ON T H E T R A N S F O R M A T I O N O F R A T E M B R Y O C E L L S O F L O W P A S S A G E BY C A R C I N O G E N I C F L U O R E N Y L H Y D R O X A M I C ACIDS AND T H E I R A C E T A T E E S T E R S H. KURZEPA, H. R. GUTMANN, D. MALEJKA-GIGANTI, A. KRAUSS, J. CERVENKA, G. J. VOSIKA, ANDR. E. RYDELL Laboratory for CancerResearch, VeteransAdministration Hospital; and the Departmentsof Biochemistry, OralPathology and Human and OralGenetics, Medicine, and Laboratory Medicine and Pathology, Universityof Minnesota, Minneapolis, Minnesota 55417
SUMMARY
Rat embryo cells of low passage subjected to a single treatment with certain carcinogenic fluorenylhydroxamic acids and their respective acetate esters showed signs of transformation in vitro, such as changes in phenotype, growth in soft agar and agglutination with concanavalin A. In addition, certain changes in karyotype and loss of diploidy were observed. There was no evidence, either by electron microscopy or by assay of RNA-dependent DNA polymerase, for the presence of virus. None of these cell lines produced tumors after inoculation into the isologous host. The results of this study lead us to suggest that malignant transformation is a multistep process and that certain criteria of transformation of rat embryo cells are associated with the initial stage(s) in which the cells are transformed without being tumorigenic. The ultimate test for malignant transformation of rat embryo cells remains the production of tumors in a susceptible host after inoculation of treated cells. Key words: transformation; rat embryo cells; fluorenylhydroxamic acids. INTRODUCTION
Investigations have been carried out in this laboratory for some time to establish a method for the screening in vitro of fluorenylamides and their activated derivatives by means of rat embryo cells. Assays in vitro have been applied to the studies of the effect of carcinogenic aromatic polycyclic hydrocarbons on hamster or mouse cell cultures [for review see Mishra and DiMayorca (1) and Heidelberger (2)]. Few reports have dealt with transformation of rat embryo cells in vitro by carcinogens in general (3-7) and specifically with transformation by carcinogenic arylamides or arylhydroxamic acids {6,8). A previous study from this laboratory has indicated that monolayers of rat embryo fibroblasts exposed to certain fluorenylhydroxamic acids and their acetates produced a high incidence of transplantable sarcomas when the cells were injected into isologous and nonisologous hosts (8k This investigation did not include quantitation of transformation. Moreover, the cells were from a commercial source and had undergone numerous passages (approximately 40) prior to multiple expos-
ures to the carcinogens. The present work was undertaken to determine whether induction of malignant transformation could be accomplished when low-passage rat embryo cells were treated with a single close of N-hydroxy-2-fluorenylacctamide (N-hydroxy-2-FAAk N-hydroxy-2-fluorenylhenzamide (N-hydroxy-2-FBA), N-hydroxy3-FAA, N-acetoxy-2-FAA, N-acetoxy-2-FBA and N-acetoxy-3-FAA. All of these compounds are potent carcinogens for the rat in vivo (9-11). Transformation in vitro was monitored by standard criteria (morphologic changes, growth in soft agar, agglutination by concanavalin A, karyotype analysis). The ultimate test for malignant transformation was the injection, into isolognus hosts, of cells derived from colonies that were judged transformed by the above criteria. MATERIALS AND METHODS
Media. Eagle's medium (MEM) containing Hanks' basal salt solution (BSS) was purchased from Microbiological Associates, Bethesda, Maryland. This medium was fortified with Lglutamine (2 mMk sodium pyruvate {1 mM), a
261
262
KURZEPA ET AL.
mixture of nonessential amino acids (0.1 mM) (Microbiological Associates, Bethesda, Md.)(12) and 10% fetal bovine serum (Grand Island Biological Co., Grand Island, N.Y.) inactivated by heating at 56 ~ C for 30 rain, and is designated fortified M E M . Cultivation of cells. Cells were obtained from 18-day-old embryos of a single Wistar-Furth rat purchased from ARS Spragne-Dawley, Madison, Wisconsin. The embryos were removed under sterile conditions, washed several times with Ca 2§ and M~§ BSS supplemented with penicillin (250 U per ml) and streptomycin (250/~g per ml) and minced. The mince was subjected to digestion by a solution of 0.25% trypsin. The digestion was carried out at 37 ~ C and repeated four times. The supernatant liquid from the first digestion was discarded. The subsequent supernataut liquids were combined and centrifuged. The resulting pellet was suspended in fortified M E M and the suspension was filtered through three layers of sterile gauze. An aliquot of the filtrate (4 ml) was delivered into 25-cm 2 flasks (Falcon Plastics, Los Angeles, Calif.) for cell propagation. The remainder was frozen in fortified M E M containing 15% glycerol and kept at - 8 5 ~ C for further use. The cells were incubated at 37 ~ C in 5% CO~:95% air (90% humidity). After 24-hr incubation of the suspension, the primary cells were rinsed with fortified M E M and replenished with the same medium. These primary cells were the source for all subsequent experiments. Compounds. N-hydroxy-2-FAA, m.p. 150151 ~ C (9); N-hydroxy-3-FAA, m.p. 131-132 ~ C (13); N-hydroxy-2-FBA, m.p. 189-191 ~ C (10); N-acetoxy-2-FAA, m.p. 111-112 ~ C (14); Nacetoxy-3-FAA, m.p. 104-105 ~ C (15); and Nacetoxy-2-FBA, m.p. 130-132 ~ C (11) were prepared by the published procedures. The compounds were dissolved in acetone (spectral grade) at a concentration of 0.5 or 1.0/~mol per ml. The acetone solutions of the hydroxamic acids or of the acetate esters were diluted 100 times with M E M or with BSS, respectively. Prior to use in the carcinogenicity tests, the stability of all compounds in M E M and BSS was tested by analysis of the medium with the use of high-pressure liquid chromatography (16,17). The recovery data indicated reasonable stability of the fluorenylhydroxamic acids in M E M during 24 hr. However, the acetate esters were not recovered from M E M indicating instability or rapid interaction with that medium. The recovery data showed greater stability of these compounds in BSS. Thus N-
acetoxy-2-FBA was recovered nearly quantitatively within 24 hr. Similarly major amounts of unchanged N-acetoxy-3-FAA were recovered within that period. However, only 10% of Nacetoxy-2-FAA was recoverable from BSS after incubation for 0.5 hr. The times of exposure of the cells to the compounds were selected according to their apparent stability. Exposure to compounds. Cells (104) of passages 5 to 7 were seeded on a plastic Petri dish with grid (60-mm diameter) and incubated for 24 hr in fortified MEM. The medium was removed and the cells were washed three times with BSS. The cells then were incubated with solutions of fluorenylhydroxamic acids and their acetates for 24 hr and 0.5 to 5 hr, respectively. Controls consisted of cells treated with M E M and M E M containing 1% acetone as well as BSS and BSS containing 1% acetone. Fifteen dishes were used for each concentration of compound and for the controls. After treatment with fluorenylhydroxamic acids, the cells were washed three times with MEM. After exposure to acetates, the cells were washed three times with BSS.
Determination of cytotoxicity and transformation. Cytotoxicity was estimated by cell count of several dishes immediately after exposure to the compounds. Cytotoxicity also was determined by plating efficiency (18). Transformation was assayed by the transformation frequency based on the number of morphologically altered colonies relative to the number of total colonies (18, 19). Morphologically altered colonies were isolated (20) and propagated in fortified M E M for further tests of transformation, such as growth in soft agar (21) and agglutination by concanavalin A (Con A) (22). For growth in soft agar, the cells from each altered colony and from the appropriate controls were grown on three to five dishes, and development of colonies was followed for 8 weeks. Agglutination was measured as described (23) except that the cells for the assay were washed with Ca 2§ and Mg2+-frec BSS and dispersed in 0.67 M phosphate buffer, pH 7.5 (10~ cells per ml). The extent of agglutination was calculated from the following expression: % A,-F agglutination ---- - - x 100, where A~ ---T,-F agglutinated cells in sample; T8 = total cells in sample; Ac = agglutinated cells in control; Tc = total cells in control; F = __At x Ts. Controls for T~ agglutination of untreated and carcinogen-treated
263
TRANSFORMATION OF RAT EMBRYO CELLS cells consisted of a cell suspension in p h o s p h a t e buffer without addition of C o n A. Test for malignancy of transformed cells. Confluent cultures derived f r o m t r a n s f o r m e d colonies a n d from the a p p r o p r i a t e controls were harvested by trypsinization with a 0.05% trypsin-0.05% E D T A solution (Microbiological Associates, Bethesda, M d . ) . After centrifugation, the ceils were w a s h e d with M E M , centrifuged a n d r e s u s p e n d e d in M E M . Approximately 107 cells per 0.1 ml M E M were injected subcutaneously into n e w b o r n W i s t a r - F u r t h rats or i n t r a m u s c u l a r l y into wean-
ling rats of the s a m e strain. T h e developed t u m o r s were excised a n d e x a m i n e d histologically. R a t s were m a i n t a i n e d on P u r i n a C h o w a n d water a d libitum for 8 m o n t h s after injection of the cells. Chromosomal analysis. N e a r - c o n f l u e u t cultures were trypsinized after exposure to colchicine for 2 to 4 hrs. T h e cells t h e n were treated with hypotonic KCI a n d subjected to fixation with acetic acid a n d methyl alcohol {1:1}. T w o sets of slides were prepared. One of these was stained with the conventional G i e m s a stain. T h e second set was treated with trypsin for G - b a n d i n g (24).
TABLE 1 TRANSFORMATIONOFR ATEMBRYOC ELLS{5THPASSAGE}AFTERAS INGLEEXPOSURETO F LUORENYLHYDROXAMIC ACIDS DETERMINED BYASSAYSIN VITROANDIN VIVO
Compound
Dose
Plating TransformaEfficiency a fion a
(nmol/ dish) (%) MEM
0
--d
Designation of Isolated Colony
Growth in AgghtinaSemisoft tion byc Agarb Con A
No. Passage Prior to Injection
Tumor Incidence
19 17 35 16
0/4 0/5 5/6 f 0/5
(%) 0e
-
-
N/7/6
-
-
Na/7/3 Na/7/4 Na/7/5
+ -
-
20 21 18 25 36
0/3 3/3 g 0/5 0/6 0/6
M E M + I % acetone
0
_d
N-Hydroxy-2-FAA
50
0.2
96-100
D'/6/1 D'/6/2 D'/6/3
4-44-4-+
++ +
16 16 24
0/6 0/5 0/12
25
0.5
90-100
D/7/4 D/7/8
+ +4-
+ -4-
15 18
0/4 0/5
25
2.0
100
C'/7/2
+'4-+
-4-
C'/7/4
-4-+
-4-
15 18 17 24 37
0/4 0/5 0/5 0/6 0/6
N-Hydroxy-2-FBA
N-hydroxy-3-FAA
0e
N/7/1 N/7/3
12
2.0
91-100
C/7/4
4-
-4-
17
1/13 h
50
1.0
97-100
A'/7/1 A'/7/6
+ +
+ 4-
22 17 26
0/5 0/5 1/6 i
25
2.0
97-100
A/7/2
4-
4-
16 34 A/7/8 4-4-4421 a Plating efficiency and transformation frequency was determined 22 days after the cells were seeded. b Symbols represent the average number of colonies on four or five Petri dishes: - : - 0-10; 4- = 10-50; 50-100; + + + = >100. c + Indicates 50% agglutination of cells. d Confluent monolayer. e No morphologic changes. f Five rats had a pleomorphic sarcoma at the site of inoculation. s Two rats had a Iibrosarcoma and one rat had a pleomorphic sarcoma at the site of inoculation. h One rat had a Wilm's tumor of kidney ~nephroblastoma). i One rat had a mammary adenocarcinoma.
0/5 0/5 0/5 ++=
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The modal number and ploidy for each cell line was established. Sets of karyotypes were prepared for detailed characterization of the chromosomal constitution of each cell line. Screening for presence of virus. Cells from untreated and carcinogen-treated cultures were examined routinely by electron microscopy for the presence of viral particles. In addition, a high speed (I00,000 x g) pellet of supernatant fluids from cultures that were subsequently inoculated into test animals was prepared and assayed in triplicate for RNA-directed DNA polymerase activity (reverse transcriptase) using the synthetic template Poly (rc) (dG)12.18 as previously described (25L
The results of transformation assays in vitro and in vivo are summarized in Table 1 and 2. No transformation was seen in the controls. An example of a control colony and a monolayer derived from this colony are shown in Fig. 1A,B, respectively. Transformation frequency by morphologic criteria (19,26,27) was almost quantitative after exposure to fluorenylhydroxamic acids. A typical picture of a transformed colony and a monolayer derived from this colony is given in Fig. 1C,D, respectively. Acetate esters produced morphologic transformation ranging from 2% to 29%, whereas no morphologic changes were noted in the controis (Table 2L There are reports in the literature that transformation of mouse embryo cells exposed to polycyclic aromatic hydrocarbons can be RESULTS graded into three classes based on the appearance Cytotoxicity as determined by cell count indi- of transformed loci. Only one of these classes is cated a 29% to 56% decrease in the number of tumorigenic (28). However, rat embryo cells that cells treated with fluorenylhydroxamic acids. The had been exposed to fluorenylhydroxamic acids corresponding decrease for cells exposed to aceand their acetates did not show morphologic altate esters was 37% to 66%. No decrease in the terations that would lend themselves to the gradanumber of cells was noted in the controls. Cytotion applied to transformed mouse cells. toxicity measured by plating efficiency 2 to 3 Variable clonal growth in semisoft agar was observed with all cells derived from colonies that weeks after exposure to the compounds ranged from 0.2 to 2% in cultures treated with fluorenyl- had been transformed morphologically by fluorhydroxamic acids (Table I} and from 1% to 5% enylhydroxamic acids and the corresponding esters (Tables 1, 2). A typical picture of a colony in cultures treated with the acetate esters (Table 2 }. Incubation of cells in M E M alone or in M E M in semisoft agar derived from cells exposed to Nhydroxy-2-FAA is seen in Fig. 2,4. In the majority containing 1% acetone showed no evidence of cytotoxicity (Table 1 ). On the other hand, incubaof cases, cells grown in control medium did not tion with BSS alone or with BSS containing 1% form colonies in soft agar. However, there were at least two colonies isolated from control medium acetone gave only plating efficiencies of 8% to which did grow in semisoft agar, although the 10% (Table 2L TABLE 2 TRANSFORMATION OF n AT EMBRYO CELLS ( 7TH P ASSAGEIAFTER A SINGLE EXPOSURE TO ACETATE ESTERS OF F LUORENYLHYDROXAMICACIDS DETERMINED BY ASSAYS IN VITRO AND IN VIVO
Compound
Dose
Plating TranslormaEfficiency a tiona
(nmol/dish) (%) BSS BSS + 1% acetone N-Acetoxy-2-FAA
Designation of Isolated Colony
No. Growthin Agglutina- Passage Semisoft tion by Prior to Agarb Con Ac Injection
Tumor Incidence
(%)
0 10 0d N/5/10/6 + 21 0 8 0d Na/0.5/10/2 _+ 22 50 4 2 E'/10/1 +++ + 25 25 5 2 E/10/1 -4-+ + 20 N-Acetoxy-2-FBA 25 1 6 F'/10/4 -4419 12 2 8 -f N-Acetoxy-3-FAA 50 1 29 B'/10/5 4-4424 25 3 7 _I a Plating efficiencyand transformation frequency was determined 13 days after the cells were seeded. b Symbols represent the average number of colonieson four or five dishes: - = 0-10; + = 10-50; + + = + + + = >100. c -4- Indicates 50% agglutination of cells. d No morphologic changes. e Two rats had a pleomorphic sarcoma at the site of inoculation. f These coloniesdid not survive subsequent propagation.
2/6 e 0/6 0/7 0/3 0/5 0/5
50-100;
TRANSFORMATION OF RAT EMBRYO CELLS
265
FIG. 1. Representative phase-contrast photographs of untreated and carcinogen-treated rat embryo cells. A, Colony of untreated cells (control). Note cells growing in a parallel-oriented pattern. B, Confluent monolayer derived from colony shown in A. C, Colony of cells treated with N-hydroxy-2-FAAas described in the text. Note loss of cellular orientation, crisscross pattern and irregular borders. D, Monolayer derived from colony shown in C. Note tendency to form multilayered loci. x150.
level of colony formation was low. Since similarly low levels of colony formation also were observed with morphologically altered colonies obtained by exposure to carcinogens, clonal growth in semisoft agar would not appear to be a reliable criterion for malignant transformation of rat cells in vitro. It seemed possible that morphologically transformed cells and bona fide tumor cells could be distinguished by quantitative differences in their growth in semisoft agar. Indeed, tumor cells derived from plcomorphic sarcomas induced in Wistar-Furth rats by subcutaneous injection of the carcinogen N-hydroxy-2-FBA exhibited generally a high colony formation in semisoft agar ranging from 500 to 11000 colonies per dish. A picture of a colony derived from tumor cells is shown in Fig. 2B. However, the variations in clonal growth between different batches of tumor cells were too large to establish a statistically significant number that would permit us to distinguish between tumorigenic and nontumorigenic cells. Moreover, there was an overlap between the lower range of the clonal growth of bona .fide
tumor cells and of the clonal growth of morphologically transformed cells that proved to be nontumorigenic by the inoculation test. As a third criterion of transformation, agglutination tests with Con A were carried out. However, no consistent pattern indicative of transformation was evident (Tables 1, 2). Thus, although the test appeared to be positive for cells that had been derived from morphologically altered colonies obtained after treatment of rat embryo cells with the carcinogens, there were also instances in which cells derived from control colonies gave 50% agglutination with Con A. Our previous data had shown that rat embryo cells that had undergone malignant transformation after multiple exposure to carcinogenic fluorenylhydroxamic acids or their corresponding acetates induced fibrosarcomas at the point of injection within 2 to 4 months after subcutaneous inoculation into isologous and nonisologous hosts (8). Accordingly, the rats injected with rat embryo cell lines in this study were observed for tumor development for as long as 8 months. Inoculation of
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FIG. 2. Clonal growth in semisoft agar. A, Colony derived from cells exposed to the carcinogen Nhydroxy-2-FAA, x200. B, Colony derived from tumor cells. >
