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Cytogenetic analysis of murine cell lines from diethylstilbestrol-induced uterine endometrial adenocarcinomas
Cytogenetic Analysis of Murine Cell Lines from Diethylstilbestrol-Induced Uterine Endometrial Adenocarcinomas Sumiyo Endo, Seiji Kodama, Retha Newbold, John McLachlan, and J. Carl Barrett ABSTRACT: Treatment of female CD-1 mice with the synthetic estrogen diethylstilbestrol (DES) on days 1 through 5 after birth results in a 90% incidence of endometrial adenocarcinomas by 18 months of age. Three cell lines were established from DES-induced uterine carcinomas and studied for specific chromosomal changes. Each cell line exhibited numerical decreases in chromosomes 9, 11, 13, and X as common abnormalities. Structural alterations involving chromosomes 3, 6, 11, and 19 occurred nonrandomly among the three cell lines. Every cell line showed a rearrangement in the long arm of chromosome 3 (3q +), a translocation between chromosomes 3 and 19 [t(3;19)], isochromosome of chromosome 11 [i(11)], and a marker chromosome (M2) either as common abnormalities or recurrent abnormalities, t(3;19), i(11), and M2 were observed also in the primary colonies from which the cell lines arose. The changes were not observed in a cell line derived from the uterus of one untreated control mouse, suggesting that these chromosomal alterations may have occurred during DES-induced neoplastic transformation. The chromosomal alterations found in the present study m a y prove useful in investigating the genetic changes involved in DES carcinogenesis.
INTRODUCTION Diethystilbestrol [DES), a synthetic estrogen and known human carcinogen, is well known to induce aneuploidy in many cell types, possibly because of its effects on microtubules [1-3]. DES induces nondisjunction in Syrian hamster embryo (SHE) cells in culture, and it has been proposed that induction of aneuploidy is involved in DES-induced cell transformation [4-7]. This conclusion is based on parallel dose-response curves for cell transformation and aneuploidy, similar cell cycle dependency for both endpoints [4, 5], correlations between the ability of DES-related compounds to induce aneuploidy and cell transformation [6], and the finding of nonrandom numerical changes in DES-induced cell lines [7]. Newbold et al. [8] have described alterations in the genital tracts of female CD-1 mice treated neonatally with DES. One of the most prominent of these alterations is endometrial adenocarcinoma, which appears in a time- and dose-
From the Laboratory of Reproductive and Developmental Toxicology (S. g., R. N., ]. McL.) and Laboratory of Molecular Carcinogenesis (S. K., ]. C. B.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triang/e Park, North Carolina. .Address reprint requests to: Dr. ]. Carl Barrett, NIGHS,MD C215, P.O. Box 12233, Research Triangle Park, NC 27709. Received July 30, 1993; accepted October 28, /993.
dependent manner after DES treatment. Ongoing research is designed to understand the relationship between the DESinduced genetic change[s) and neoplastic transformation in this animal model. Using the techniques of dissociation of uteri with collagenase and culturing cells on a feeder cell matrix, three tumorigenic cell lines derived from the uterine endometrium of CD-1 mice treated neonatally with DES were established in a previous study [9]. In these cell lines no mutations were found [9] in several genes that have previously been shown to be associated with human endometrial carcinomas [10-12]. To further analyze these tumors, cytogenetic analyses of these three cell lines were performed in order to understand possible genetic events involved in DES-induced carcinogenesis. MATERIALS AND METHODS
Cell Lines Cell lines 091488, 092188, and 092788 were derived from uteri of 19-month-old CD-1 female mice treated neonatally with DES as previously described [8]. The cell line 021889, C1-2, was derived from the uterus of a female mouse treated neonatally with corn oil (control). Methods for establishing the cell lines were described previously [9].
Cytogenetics Primary transformed colonies were trypsinized by holding 99
© 1994 Elsevier Science Inc.
Cancer Genet Cytogenet 74:99-103 (1994)
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100
S. E n d o et al.
Table 1
C y t o g e n e t i c f i n d i n g s i n m u r i n e cell l i n e s f r o m D E S - i n d u c e d u t e r i n e endometrial adenocarcinoma
Cell line
Passage
Modal chromosome number
091488 a
10
70-86
092188 a
10
Common chromosome abnormalities
Recurrent chromosome abnormalities
( - X , - X , - X ) , - 1, - 2, - 4, + 5, - 8, - 9, - 11,12,1 3 , - 1 4 , + 1 5 , - 1 8 , + 19
-6,+6,-
3q +, + t(1;?), + t(6;?), + t(3;10), + t(3;19), + M1
+ dup(1}, + i{1), + t(10;19), + i(11), + t(11;?), + t(2;?), + t{3;?), + M2, + M3
68-81
- 1, - 9,-
7,-10
-2,-2,+3,+3,-4,-4,+5,+6,
11, - 13, - X
-7,-8,
(-10,-10,-10),-12,
( - 14, - 14, - 14), + 15, - 16, - 18 +
092788 a
10
68-84
3q +, + t(4;6), + t(9;X), + i(11), + i(13), + t(6;13), + t(19;?), + t(13;?), + M2, + M4, + M5
t(3;19)
- 2, - 4 , - 5, - 7, - 8, - 9, - 11, - 12, - 13,-
-
1,
-
3,
+ 3,
+ 6,
+ 6,
-
10,
-
15,
+ 15, - 16, - 19
14, - 18, -X
+ t(4;6), + t(3;19), + M4
+ dup(1), + i(1), + t(1;2),3q + ,
+ i(4), + i(5), + i(6), + t(6;?), + i(11), + t(15;X), + t(6;16), + t(15;16), t(5;19), + i(X), + t(13;?), + t(19;?), +M2, +M6 Abbreviations: +, gain of a whole chromosome; - , loss of a whole chromosome; q +, rearrangement of a long arm of a chromosome; t. translocation; i, isochromosome; M, marker chromosome.
a Deviation from tetraploidy.
t h e d i s h at a n a n g l e so t h a t o n l y a p o r t i o n of t h e c o l o n i e s c a m e i n c o n t a c t w i t h t h e t r y p s i n s o l u t i o n (0.25% t r y p s i n , 0.02 % EDTA). A f t e r 2 - 5 m i n u t e s o f t r y p s i n e x p o s u r e at r o o m t e m p e r a t u r e , t h e d i s s o c i a t e d c e l l s w e r e r e m o v e d by p i p e t ting w i t h a Pasteur p i p e t , r e s u s p e n d e d i n t h e c u l t u r e m e d i u m (M5 f o r m u l a t i o n [9]), a n d t r a n s f e r r e d into 2 5 - c m 2 flasks for e s t a b l i s h m e n t of cell lines. T h e p o r t i o n of t h e c o l o n i e s t h a t r e m a i n e d a t t a c h e d to t h e d i s h w a s r e f e d w i t h m e d i u m a n d u s e d for c y t o g e n e t i c a n a l y s i s 2 4 - 4 8 h o u r s later. For c e l l lines, flasks c o n t a i n i n g l o g a r i t h m i c a l l y d i v i d i n g c e l l s w e r e t r e a t e d w i t h C o l c e m i d (final c o n c e n t r a t i o n , 0.05 ~g/ml) i n M5 m e d i u m for I h o u r at 37°C. For p r i m a r y t r a n s f o r m e d c o l o n i e s , c e l l s i n intact c o l o n i e s w e r e t r e a t e d w i t h
Table 2
C o l c e m i d (final c o n c e n t r a t i o n , 0.05 ~g/ml) for 2 h o u r s at 37°C. After t r e a t m e n t w i t h C o l c e m i d , cells w e r e d e t a c h e d by 0.25% t r y p s i n a n d 0.02 % EDTA, c o l l e c t e d b y c e n t r i f u g a t i o n , a n d r e s u s p e n d e d i n 0.075 M KC1 for 2 0 - 3 0 m i n u t e s at r o o m t e m perature. After this h y p o t o n i c t r e a t m e n t , cells w e r e fixed w i t h m e t h a n o l : g l a c i a l acetic a c i d (3:1, v/v), a n d a i r - d r i e d c h r o mosome slides were prepared. Karyotyping C h r o m o s o m e s w e r e b a n d e d a c c o r d i n g to a q u i n a c r i n e f l u o r e s c e n c e b a n d i n g m e t h o d [13]. C h r o m o s o m e i d e n t i f i c a t i o n w a s p e r f o r m e d a c c o r d i n g to t h e s t a n d a r d i z e d n o m e n c l a t u r e for M u s m u s c u l u s [14]. R e c u r r e n t c h r o m o s o m a l ab-
C h r o m o s o m e s i n v o l v e d i n n u m e r i c a l a n d / o r s t r u c t u r a l c h a n g e s i n m u r i n e cell l i n e s f r o m D E S - i n d u c e d uterine endometrial adenocarcinoma Chromosome number
Cell line 091488
1
2
3
4
5
-
-
+
-
+
6 ( -
7 )
8
9
10
11
(7)
-
-
( -
q+
(q+)
(-)
(-)
-
(-)
-
)
.
12 .
.
13
14
.
15
16
+
17
18 -
19 +
X -
+
092188
092788
q+
(q+)
q+
-
(-)
+
(-)
q+ (-) + q+
(q+) -
(-)
-
(q+)
(q+}
q+
q+
(+)
(+)
(q+) + (q+)
q+
-
(q+) -
C- )
(q+) . . (q+)
q+ (-)
-
(-)
(+)
(-)
(q+) . .
(- }
{- )
(q+)
(q+)
{q+)
(-)
-
+
-
q+ (q+) (- } + q+ (q+)
Abbreviations: +, gain of a whole chromosome; +, loss of a whole chromosome; q +, rearrangement of a long arm of a chromosome; (), recurrent abnormali-
ties; no parenthesis, common abnormalities.
Karyotypes of DES-Induced Endometrial Adenocarcinomas normalities (observed in at least 20% of metaphases) and c o m m o n chromosomal abnormalities (observed in at least 80% of metaphases) were noted. For each cell line, chromosomal analysis consisted of 10 banded karyotypes and at least 40 metaphases were analyzed under the fluorescence microscope. If chromosomal changes were found in the cell lines, those were analyzed in at least 50 metaphase spreads from their primary colonies.
RESULTS Cytogenetic Findings in Murine Cell Lines from DESInduced Uterine Endometrial Adenocarcinomas Numerical Changes. All three cell lines at passage 10 were near-tetraploid with modal chromosome numbers ranging from 68 to 86 (Table 1). Chromosomal losses and gains indicated in Table 1 are relative to tetraploid cells (80 chromosomes). Every cell exhibited numerical decreases in chromosomes 9, 11, 13, and X as c o m m o n abnormalities (Tables I and 2). Gains of chromosome 6 and losses of chromosome 10 were found as recurrent abnormalities in every cell line (Tables I and 2). Numerical gains of chromosomes 3, 6, and 19 were observed in all cell lines due to RJabertsonian translocations. Structural Changes. Nonrandom structural changes involving chromosomes 3, 6, 11, or 19 were observed in all three cell lines at passage 10. All translocations involving chromosome 3 were Robertsonian translocations. The translocation between chromosomes 3 and 19 [t(3:19)] was found as a c o m m o n abnormality in every cell line (Table 1) (Fig. 1). In each of the cell lines, all the cells had more than one t(3;19) chromosome. The rearrangement 3q + involving a breakpoint in the distal region of the long arm of chromosome 3 was found as either a c o m m o n or recurrent abnormality in all three cell lines (Table 1). Cell line 091488 exhibited chromosome 3q + as a c o m m o n abnormality, and the other two lines exhibited it as a recurrent abnormality. Two other translocations of chromosome 3, t(3;10) and t(3;?), were found as a c o m m o n abnormality and a recurrent abnormality, respectively, in cell line 091488 only. Four different translocations of chromosome 6, i.e., t(4;6), t(6;?), t(6;13), and t(6;16), occurred as c o m m o n abnormalities or recurrent abnormalities among the three cell lines (Table 1). The breakpoint in t(6;?) was in a distal part of chromosome 6 and that in t(4;6) was in a proximal part of chromosome 6. The other alterations were Robertsonian translocations. Isochromosome 6, i(6), was a recurrent abnormality only in cell line 092788. Isochromosome 11, i(11), was observed as a recurrent abnormality in all three cell lines (Fig. 1). A Robertsonian translocation involving chromosome 11, t(11;?) was a recurrent abnormality in cell line 091488. In addition to t(3;19), chromosome 19 contributed to two other translocations, t(10;19) and t(19;?), which were found recurrently in cell lines 091488 or 092188, respectively. All three cell lines contained a specific, small metacentric marker chromosome (M2) (Fig. 1) that was found as a recurrent abnormality.
101 Double minutes (drain) were observed frequently (> 80% of cells analyzed} in each cell line. The numbers of dmin chromosomes in a given cell varied between 1 and 100.
Cytogenetic Findings in P r i m a r y Trang~ormed Colonies From Which the DES-cell Lines Were Derived All cell lines exhibited a t(3;19) chromosome as a c o m m o n abnormality and i(11) and M2 chromosomes as recurrent abnormalities. To determine whether or not these chromosome changes occurred in the primary transformed colonies from which the cell lines arose, karyotypes of the primary transformed colonies were analyzed. Primary colonies of all three cell lines were aneuploid, with a modal chromosome number (68-81) similar to that of the cell lines, t(3;19) was observed in more than 95% of the cells analyzed in 091488 and 092788 primary colonies. We could not analyze as many metaphases in 092188 primary colonies; however, 15 of 17 cells observed had t(3;19). i(11) was found recurrently both in 091488 (55% of the analyzed cells) and 092788 (64% of the analyzed cells) in primary colonies. In 092188 primary colonies, seven of 17 cells showed i(11). A substantial population of 091488 primary colonies (57%) exhibited an M2 chromosome, which was observed in only 12% of cells from 092788 primary colonies. Only one of 17 cells examined had the M2 chromosome in 092188 primary colonies. Double minutes frequently were found also in every primary colony. The cells had variable numbers of dmin chromosomes ranging from I to 100, as was seen in the cell lines.
Cytogenetic Findings in the Murine Cell Line 021889, C1-2 from Control Uterine Tissue A control cell line, 021889, C1-2, was derived from a single uterus treated neonatally with corn oil. This cell line was nontumorigenic and was characterized by a fibroblastic morphology that was in contrast to the three cell lines obtained from DES-treated uteri, which formed tumors u p o n injection into nude mice. The modal chromosome number was between 75 and 80 in the primary colonies as well as the cell lines at passage 3. No t(3;19), i(11), and M2 could be found in the primary colonies or in the cell lines at passage 3. Neither the primary colonies nor the cell lines at passage 3 contained DM chromosomes. DISCUSSION We observed nonrandom structural alterations involving chromosomes 3, 6, 11, and 19 among the three tumorigenic cell lines at passage 10 derived from DES-induced adenocarcinemas. There were four types of structural alterations involving chromosomes 3, 3q+, t(3;19), t(3;10), and t(3;?). Chromosomes 3q+ and t(3;19) occurred in all three cell lines. t(3;19), especially, occurred as a c o m m o n abnormality in every cell line, even at the early passages. It is unlikely that this change occurred during cell culture because the primary transformed colonies showed this nourandom translocation. Because the translocations observed are formed by Robertsonian fusions, no genes on chromosome 3 may be lost or rearranged. However, the occurrence of these translocations
0
X
T m
T m
0
L~
Lf ) ~w
0
C~ T m
v
~D Tm V
~D
X
Karyotypes of DES-Induced Endometrial A d e n o c a r c i n o m a s results in gains of c h r o m o s o m e 3. Because the N-RAS protooncogene has been reported to be located on chromosome 3 [15], ongoing experiments in our laboratory are examining the role of N-RAS in the transformation of the cells. We found five types of structural alterations involving c h r o m o s o m e 6: t(6;?), t(4;6), t(6;13), t(6;16), and i(6). Sometimes the same alterations were found in different cell lines. In all cases, alterations in c h r o m o s o m e 6 resulted in gains of chromosome 6. The K-RAS-2, RAF-1, RET, and MET oncogenes are located on c h r o m o s o m e 6 [15, 16]. We did not find any point mutation or amplification in K-RAS-2 in a previous study [9], and genetic alterations in RAF-1, RET, and MET are u n d e r study. Isochromosome 11, w h i c h was observed recurrently in all three cell lines, may have occurred by centric fusion and, if so, i(11) is genetically equivalent to disomy 11. However, losses of c h r o m o s o m e 11 occurred frequently also in every cell line. On mouse chromosome 11, the genes for the ERB-B oncogene, PKC (protein kinase C), THRAI (thyroid hormone receptor a) COLA-1 (procollagen type I, alpha 1), KRT-1 (keratin gene complex-I), and P53, NF1, and BRCA1, t u m o r suppressor genes are located [15]. P r e l i m i n a r y data suggest that P53 is not overexpressed in these cell lines. The observed alterations in chromosome 19, t(3;19), t(10;19), and t(19;?), result in gains of chromosome 19. It s h o u l d be noted that Robertsonian translocations result in gains of chromosomes 3, 6, and 19. Because it is suggested that a gain of a specific chromosome, such as a trisomy of chromosome 11, is strongly correlated with DES-induced cell transformation in SHE cells, the increased copies of chromosomes 3, 6, and 19 may have a significant role in DESinduced carcinogenesis in mice. We also observed drain chromosomes in all three cell lines. Double minute chromosomes frequently represent cytologic forms of amplified genes [17, 19]. Our previous study demonstrated a three- to fourfold amplification of the c-myc gene only in the 092188 cell line at passage 10 [9]. In the 092188 cell line at passage 10, one recurrent a b n o r m a l i t y was a gain of chromosome 15, on w h i c h the c-myc gene is located [15, 18]. Interestingly, the 091488 cell line showed a gain of chromosome 15 c o m m o n l y at passage 10. However, we could not detect elevated expression of c-myc in the 091488 cell line at passages 16-18 [9]. The existence of m u l t i p l e drain chromosomes suggests amplification of other genes in all three cell lines. The n o n r a n d o m c h r o m o s o m e changes observed in the present study could not be found in the 021889, C1-2 control line, w h i c h was derived from the uterus of a control animal. We also failed to observe any drain chromosomes in this cell line or in its p r i m a r y colonies. Thus, the n o n r a n d o m c h r o m o s o m e changes were specific for DES-induced neoplastic transformation. These n o n r a n d o m chromosome alterations may provide clues for the critical genetic changes involved in DES-induced carcinogenesis. REFERENCES 1. Sato Y, Mumi T, Tsumumya M, Saito H, Kodama M (1984): Dis-
103
2.
3.
4.
5.
6.
7.
8.
ruptive effect of diethylstilbestrol on microtubules. Gann 75:1046-1048. Sharp DC, Parry JM {1985): Diethylstilbestrol: the binding and effects of diethylstilbestrolupon the polymerization and depolymerization of purified microtubule protein in vitro. Carcinogenesis 6:865-871. Tucker RW, Barrett JC [1986): Decreased numbers of spindle and cytoplasmic microtubules in hamster embryo cells treated with a carcinogen, diethylstilbestrol. Cancer Res 42:2088-2095. Tsutsui T, Maizumi H, McLachlan JA, Barrett JC (1983): Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosome mechanism in carcinogenesis. Cancer Res 43:3814-3821. Barrett JC, Hesterberg TW, Oshimura M, Tsutsui T {1985): Role of chemically induced mutagenic events in neoplastic transformation of Syrian hamster embryo cells. In: Carcinogenesis, Vol. 9, Barrett JC, Tennant RW, eds. Raven Press, NY, pp. 123-137. Tsutsui T, Suzuki N, Fukuda S, Sato M, Maizumi H, McLachlan JA, Barrett JC {1987): 17~l-estradiol-induced cell transformation and aneuploidy of Syrian hamster embryo cells in culture. Carcinogenesis 8:1715-1719. Ozawa N, Oshimura M, McLachlan JA, Barrett JC {1989]: Nonrandom karyotypic changes in immortal and tumorigenic Syrian hamster cells induced by diethylstilbestrol.Cancer Genet Cytogenet 38:271-282. Newbold RR, Bullock BC, McLachlan JA (1990): Uterine adenocarcinoma in mice following developmental treatment with estrogens: A model for hormonal carcinogenesis. Cancer Res 50:7677-7681.
9. Hdbert CD, Endo S, Korach KS, Boyd J, Barrett JC, McLachlan JA, Newbold RR {1991): Characterization of murine cell lines from diethylstilbestrol-induceduterine endometrial adenocarcinomas. In Vitro Cell Dev Biol 28A:327-336. 10. Boyd J, Risinger JI, Walmer DK, Cho KR, Vogelstein B, Barrett JC (1991): Altered structure and expression of the DCC gene in human endometrial carcinoma. Proc Amer Assoc Cancer Res 32:209. 11. Enomoto T, Inoue M, Perantoni AO, Terakawa N, Tanizawa O, Rice JM (1990): K-ras activation in neoplasms of the human female reproductive tract. Cancer Res 50:6139-6145. 12. Boyd J, Risinger JI (1991): Analysis of oncogene alterations in human endometrial carcinoma: prevalence ofras mutations. Mol Carcinog 4:189-195. 13. Casperson T, Lomakka G, Zech L {1971): The 24 fluorescence patterns of the human metaphase chromosomes: distinguishing characters and variability. Hereditas 67:89-102. 14. Nesbitt MN, Francke U {1973]:A system of nomenclature for band patterns of mouse chromosomes. Chromosoma 41:145-158. 15. O'Brien SJ, Womack JE, Lyons LA, Moore KJ, Jenkins NA, Cop eland NG {1993): Anchored reference loci for comparative genome mapping in mammals. Nature C-enet 3:103-111. 16. Sakaguchi AY, Lalley PA, Zabel BU, Ellis RW, Scolnick EM, Naylor SL [1984): Chromosome assignments of four mouse cellular homologs of sarcoma and leukemia virus oncogenes. Proc Natl Acad Sci USA 81:525-529. 17. Cowell JK (1982): Double minutes and homogeneously staining regions: gene amplification in mammalian cells. Annu Rev Genet 16:21-59. 18. Sakaguchi AY, Lalley PA, Naylor SL (1983): Human and mouse cellular myc protooncogenes reside on chromosomes involved in numerical and structural aberrations in cancer. Somatic Cell Genet 9:391-405. 19. Nagl W {1990]: Gene amplification and related events. Biotechnology in Agriculture and Forestry 11:153-201.
INTRODUCTION Diethystilbestrol [DES), a synthetic estrogen and known human carcinogen, is well known to induce aneuploidy in many cell types, possibly because of its effects on microtubules [1-3]. DES induces nondisjunction in Syrian hamster embryo (SHE) cells in culture, and it has been proposed that induction of aneuploidy is involved in DES-induced cell transformation [4-7]. This conclusion is based on parallel dose-response curves for cell transformation and aneuploidy, similar cell cycle dependency for both endpoints [4, 5], correlations between the ability of DES-related compounds to induce aneuploidy and cell transformation [6], and the finding of nonrandom numerical changes in DES-induced cell lines [7]. Newbold et al. [8] have described alterations in the genital tracts of female CD-1 mice treated neonatally with DES. One of the most prominent of these alterations is endometrial adenocarcinoma, which appears in a time- and dose-
From the Laboratory of Reproductive and Developmental Toxicology (S. g., R. N., ]. McL.) and Laboratory of Molecular Carcinogenesis (S. K., ]. C. B.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triang/e Park, North Carolina. .Address reprint requests to: Dr. ]. Carl Barrett, NIGHS,MD C215, P.O. Box 12233, Research Triangle Park, NC 27709. Received July 30, 1993; accepted October 28, /993.
dependent manner after DES treatment. Ongoing research is designed to understand the relationship between the DESinduced genetic change[s) and neoplastic transformation in this animal model. Using the techniques of dissociation of uteri with collagenase and culturing cells on a feeder cell matrix, three tumorigenic cell lines derived from the uterine endometrium of CD-1 mice treated neonatally with DES were established in a previous study [9]. In these cell lines no mutations were found [9] in several genes that have previously been shown to be associated with human endometrial carcinomas [10-12]. To further analyze these tumors, cytogenetic analyses of these three cell lines were performed in order to understand possible genetic events involved in DES-induced carcinogenesis. MATERIALS AND METHODS
Cell Lines Cell lines 091488, 092188, and 092788 were derived from uteri of 19-month-old CD-1 female mice treated neonatally with DES as previously described [8]. The cell line 021889, C1-2, was derived from the uterus of a female mouse treated neonatally with corn oil (control). Methods for establishing the cell lines were described previously [9].
Cytogenetics Primary transformed colonies were trypsinized by holding 99
© 1994 Elsevier Science Inc.
Cancer Genet Cytogenet 74:99-103 (1994)
655 A v e n u e of the Americas, N e w York, NY 10010
0165-4608/94/$07.00
100
S. E n d o et al.
Table 1
C y t o g e n e t i c f i n d i n g s i n m u r i n e cell l i n e s f r o m D E S - i n d u c e d u t e r i n e endometrial adenocarcinoma
Cell line
Passage
Modal chromosome number
091488 a
10
70-86
092188 a
10
Common chromosome abnormalities
Recurrent chromosome abnormalities
( - X , - X , - X ) , - 1, - 2, - 4, + 5, - 8, - 9, - 11,12,1 3 , - 1 4 , + 1 5 , - 1 8 , + 19
-6,+6,-
3q +, + t(1;?), + t(6;?), + t(3;10), + t(3;19), + M1
+ dup(1}, + i{1), + t(10;19), + i(11), + t(11;?), + t(2;?), + t{3;?), + M2, + M3
68-81
- 1, - 9,-
7,-10
-2,-2,+3,+3,-4,-4,+5,+6,
11, - 13, - X
-7,-8,
(-10,-10,-10),-12,
( - 14, - 14, - 14), + 15, - 16, - 18 +
092788 a
10
68-84
3q +, + t(4;6), + t(9;X), + i(11), + i(13), + t(6;13), + t(19;?), + t(13;?), + M2, + M4, + M5
t(3;19)
- 2, - 4 , - 5, - 7, - 8, - 9, - 11, - 12, - 13,-
-
1,
-
3,
+ 3,
+ 6,
+ 6,
-
10,
-
15,
+ 15, - 16, - 19
14, - 18, -X
+ t(4;6), + t(3;19), + M4
+ dup(1), + i(1), + t(1;2),3q + ,
+ i(4), + i(5), + i(6), + t(6;?), + i(11), + t(15;X), + t(6;16), + t(15;16), t(5;19), + i(X), + t(13;?), + t(19;?), +M2, +M6 Abbreviations: +, gain of a whole chromosome; - , loss of a whole chromosome; q +, rearrangement of a long arm of a chromosome; t. translocation; i, isochromosome; M, marker chromosome.
a Deviation from tetraploidy.
t h e d i s h at a n a n g l e so t h a t o n l y a p o r t i o n of t h e c o l o n i e s c a m e i n c o n t a c t w i t h t h e t r y p s i n s o l u t i o n (0.25% t r y p s i n , 0.02 % EDTA). A f t e r 2 - 5 m i n u t e s o f t r y p s i n e x p o s u r e at r o o m t e m p e r a t u r e , t h e d i s s o c i a t e d c e l l s w e r e r e m o v e d by p i p e t ting w i t h a Pasteur p i p e t , r e s u s p e n d e d i n t h e c u l t u r e m e d i u m (M5 f o r m u l a t i o n [9]), a n d t r a n s f e r r e d into 2 5 - c m 2 flasks for e s t a b l i s h m e n t of cell lines. T h e p o r t i o n of t h e c o l o n i e s t h a t r e m a i n e d a t t a c h e d to t h e d i s h w a s r e f e d w i t h m e d i u m a n d u s e d for c y t o g e n e t i c a n a l y s i s 2 4 - 4 8 h o u r s later. For c e l l lines, flasks c o n t a i n i n g l o g a r i t h m i c a l l y d i v i d i n g c e l l s w e r e t r e a t e d w i t h C o l c e m i d (final c o n c e n t r a t i o n , 0.05 ~g/ml) i n M5 m e d i u m for I h o u r at 37°C. For p r i m a r y t r a n s f o r m e d c o l o n i e s , c e l l s i n intact c o l o n i e s w e r e t r e a t e d w i t h
Table 2
C o l c e m i d (final c o n c e n t r a t i o n , 0.05 ~g/ml) for 2 h o u r s at 37°C. After t r e a t m e n t w i t h C o l c e m i d , cells w e r e d e t a c h e d by 0.25% t r y p s i n a n d 0.02 % EDTA, c o l l e c t e d b y c e n t r i f u g a t i o n , a n d r e s u s p e n d e d i n 0.075 M KC1 for 2 0 - 3 0 m i n u t e s at r o o m t e m perature. After this h y p o t o n i c t r e a t m e n t , cells w e r e fixed w i t h m e t h a n o l : g l a c i a l acetic a c i d (3:1, v/v), a n d a i r - d r i e d c h r o mosome slides were prepared. Karyotyping C h r o m o s o m e s w e r e b a n d e d a c c o r d i n g to a q u i n a c r i n e f l u o r e s c e n c e b a n d i n g m e t h o d [13]. C h r o m o s o m e i d e n t i f i c a t i o n w a s p e r f o r m e d a c c o r d i n g to t h e s t a n d a r d i z e d n o m e n c l a t u r e for M u s m u s c u l u s [14]. R e c u r r e n t c h r o m o s o m a l ab-
C h r o m o s o m e s i n v o l v e d i n n u m e r i c a l a n d / o r s t r u c t u r a l c h a n g e s i n m u r i n e cell l i n e s f r o m D E S - i n d u c e d uterine endometrial adenocarcinoma Chromosome number
Cell line 091488
1
2
3
4
5
-
-
+
-
+
6 ( -
7 )
8
9
10
11
(7)
-
-
( -
q+
(q+)
(-)
(-)
-
(-)
-
)
.
12 .
.
13
14
.
15
16
+
17
18 -
19 +
X -
+
092188
092788
q+
(q+)
q+
-
(-)
+
(-)
q+ (-) + q+
(q+) -
(-)
-
(q+)
(q+}
q+
q+
(+)
(+)
(q+) + (q+)
q+
-
(q+) -
C- )
(q+) . . (q+)
q+ (-)
-
(-)
(+)
(-)
(q+) . .
(- }
{- )
(q+)
(q+)
{q+)
(-)
-
+
-
q+ (q+) (- } + q+ (q+)
Abbreviations: +, gain of a whole chromosome; +, loss of a whole chromosome; q +, rearrangement of a long arm of a chromosome; (), recurrent abnormali-
ties; no parenthesis, common abnormalities.
Karyotypes of DES-Induced Endometrial Adenocarcinomas normalities (observed in at least 20% of metaphases) and c o m m o n chromosomal abnormalities (observed in at least 80% of metaphases) were noted. For each cell line, chromosomal analysis consisted of 10 banded karyotypes and at least 40 metaphases were analyzed under the fluorescence microscope. If chromosomal changes were found in the cell lines, those were analyzed in at least 50 metaphase spreads from their primary colonies.
RESULTS Cytogenetic Findings in Murine Cell Lines from DESInduced Uterine Endometrial Adenocarcinomas Numerical Changes. All three cell lines at passage 10 were near-tetraploid with modal chromosome numbers ranging from 68 to 86 (Table 1). Chromosomal losses and gains indicated in Table 1 are relative to tetraploid cells (80 chromosomes). Every cell exhibited numerical decreases in chromosomes 9, 11, 13, and X as c o m m o n abnormalities (Tables I and 2). Gains of chromosome 6 and losses of chromosome 10 were found as recurrent abnormalities in every cell line (Tables I and 2). Numerical gains of chromosomes 3, 6, and 19 were observed in all cell lines due to RJabertsonian translocations. Structural Changes. Nonrandom structural changes involving chromosomes 3, 6, 11, or 19 were observed in all three cell lines at passage 10. All translocations involving chromosome 3 were Robertsonian translocations. The translocation between chromosomes 3 and 19 [t(3:19)] was found as a c o m m o n abnormality in every cell line (Table 1) (Fig. 1). In each of the cell lines, all the cells had more than one t(3;19) chromosome. The rearrangement 3q + involving a breakpoint in the distal region of the long arm of chromosome 3 was found as either a c o m m o n or recurrent abnormality in all three cell lines (Table 1). Cell line 091488 exhibited chromosome 3q + as a c o m m o n abnormality, and the other two lines exhibited it as a recurrent abnormality. Two other translocations of chromosome 3, t(3;10) and t(3;?), were found as a c o m m o n abnormality and a recurrent abnormality, respectively, in cell line 091488 only. Four different translocations of chromosome 6, i.e., t(4;6), t(6;?), t(6;13), and t(6;16), occurred as c o m m o n abnormalities or recurrent abnormalities among the three cell lines (Table 1). The breakpoint in t(6;?) was in a distal part of chromosome 6 and that in t(4;6) was in a proximal part of chromosome 6. The other alterations were Robertsonian translocations. Isochromosome 6, i(6), was a recurrent abnormality only in cell line 092788. Isochromosome 11, i(11), was observed as a recurrent abnormality in all three cell lines (Fig. 1). A Robertsonian translocation involving chromosome 11, t(11;?) was a recurrent abnormality in cell line 091488. In addition to t(3;19), chromosome 19 contributed to two other translocations, t(10;19) and t(19;?), which were found recurrently in cell lines 091488 or 092188, respectively. All three cell lines contained a specific, small metacentric marker chromosome (M2) (Fig. 1) that was found as a recurrent abnormality.
101 Double minutes (drain) were observed frequently (> 80% of cells analyzed} in each cell line. The numbers of dmin chromosomes in a given cell varied between 1 and 100.
Cytogenetic Findings in P r i m a r y Trang~ormed Colonies From Which the DES-cell Lines Were Derived All cell lines exhibited a t(3;19) chromosome as a c o m m o n abnormality and i(11) and M2 chromosomes as recurrent abnormalities. To determine whether or not these chromosome changes occurred in the primary transformed colonies from which the cell lines arose, karyotypes of the primary transformed colonies were analyzed. Primary colonies of all three cell lines were aneuploid, with a modal chromosome number (68-81) similar to that of the cell lines, t(3;19) was observed in more than 95% of the cells analyzed in 091488 and 092788 primary colonies. We could not analyze as many metaphases in 092188 primary colonies; however, 15 of 17 cells observed had t(3;19). i(11) was found recurrently both in 091488 (55% of the analyzed cells) and 092788 (64% of the analyzed cells) in primary colonies. In 092188 primary colonies, seven of 17 cells showed i(11). A substantial population of 091488 primary colonies (57%) exhibited an M2 chromosome, which was observed in only 12% of cells from 092788 primary colonies. Only one of 17 cells examined had the M2 chromosome in 092188 primary colonies. Double minutes frequently were found also in every primary colony. The cells had variable numbers of dmin chromosomes ranging from I to 100, as was seen in the cell lines.
Cytogenetic Findings in the Murine Cell Line 021889, C1-2 from Control Uterine Tissue A control cell line, 021889, C1-2, was derived from a single uterus treated neonatally with corn oil. This cell line was nontumorigenic and was characterized by a fibroblastic morphology that was in contrast to the three cell lines obtained from DES-treated uteri, which formed tumors u p o n injection into nude mice. The modal chromosome number was between 75 and 80 in the primary colonies as well as the cell lines at passage 3. No t(3;19), i(11), and M2 could be found in the primary colonies or in the cell lines at passage 3. Neither the primary colonies nor the cell lines at passage 3 contained DM chromosomes. DISCUSSION We observed nonrandom structural alterations involving chromosomes 3, 6, 11, and 19 among the three tumorigenic cell lines at passage 10 derived from DES-induced adenocarcinemas. There were four types of structural alterations involving chromosomes 3, 3q+, t(3;19), t(3;10), and t(3;?). Chromosomes 3q+ and t(3;19) occurred in all three cell lines. t(3;19), especially, occurred as a c o m m o n abnormality in every cell line, even at the early passages. It is unlikely that this change occurred during cell culture because the primary transformed colonies showed this nourandom translocation. Because the translocations observed are formed by Robertsonian fusions, no genes on chromosome 3 may be lost or rearranged. However, the occurrence of these translocations
0
X
T m
T m
0
L~
Lf ) ~w
0
C~ T m
v
~D Tm V
~D
X
Karyotypes of DES-Induced Endometrial A d e n o c a r c i n o m a s results in gains of c h r o m o s o m e 3. Because the N-RAS protooncogene has been reported to be located on chromosome 3 [15], ongoing experiments in our laboratory are examining the role of N-RAS in the transformation of the cells. We found five types of structural alterations involving c h r o m o s o m e 6: t(6;?), t(4;6), t(6;13), t(6;16), and i(6). Sometimes the same alterations were found in different cell lines. In all cases, alterations in c h r o m o s o m e 6 resulted in gains of chromosome 6. The K-RAS-2, RAF-1, RET, and MET oncogenes are located on c h r o m o s o m e 6 [15, 16]. We did not find any point mutation or amplification in K-RAS-2 in a previous study [9], and genetic alterations in RAF-1, RET, and MET are u n d e r study. Isochromosome 11, w h i c h was observed recurrently in all three cell lines, may have occurred by centric fusion and, if so, i(11) is genetically equivalent to disomy 11. However, losses of c h r o m o s o m e 11 occurred frequently also in every cell line. On mouse chromosome 11, the genes for the ERB-B oncogene, PKC (protein kinase C), THRAI (thyroid hormone receptor a) COLA-1 (procollagen type I, alpha 1), KRT-1 (keratin gene complex-I), and P53, NF1, and BRCA1, t u m o r suppressor genes are located [15]. P r e l i m i n a r y data suggest that P53 is not overexpressed in these cell lines. The observed alterations in chromosome 19, t(3;19), t(10;19), and t(19;?), result in gains of chromosome 19. It s h o u l d be noted that Robertsonian translocations result in gains of chromosomes 3, 6, and 19. Because it is suggested that a gain of a specific chromosome, such as a trisomy of chromosome 11, is strongly correlated with DES-induced cell transformation in SHE cells, the increased copies of chromosomes 3, 6, and 19 may have a significant role in DESinduced carcinogenesis in mice. We also observed drain chromosomes in all three cell lines. Double minute chromosomes frequently represent cytologic forms of amplified genes [17, 19]. Our previous study demonstrated a three- to fourfold amplification of the c-myc gene only in the 092188 cell line at passage 10 [9]. In the 092188 cell line at passage 10, one recurrent a b n o r m a l i t y was a gain of chromosome 15, on w h i c h the c-myc gene is located [15, 18]. Interestingly, the 091488 cell line showed a gain of chromosome 15 c o m m o n l y at passage 10. However, we could not detect elevated expression of c-myc in the 091488 cell line at passages 16-18 [9]. The existence of m u l t i p l e drain chromosomes suggests amplification of other genes in all three cell lines. The n o n r a n d o m c h r o m o s o m e changes observed in the present study could not be found in the 021889, C1-2 control line, w h i c h was derived from the uterus of a control animal. We also failed to observe any drain chromosomes in this cell line or in its p r i m a r y colonies. Thus, the n o n r a n d o m c h r o m o s o m e changes were specific for DES-induced neoplastic transformation. These n o n r a n d o m chromosome alterations may provide clues for the critical genetic changes involved in DES-induced carcinogenesis. REFERENCES 1. Sato Y, Mumi T, Tsumumya M, Saito H, Kodama M (1984): Dis-
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7.
8.
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