Karyotypic Characterization of Established Cell Lines Derived from a Squamous Cell Carcinoma and an Adenocarcinoma of Human Lung Cancers Martin Erdel, Wolfgang Peter, Eberhard Spiess, G~inter Trefz, and Werner Ebert
ABSTRACT: Two non-small cell carcinoma cell lines from the major histopathologic groups of human lung cancers have been karyotyped: HS-24 was established from a squamous cell carcinoma, and SB-3 was obtained from a metastasis of a poorly differentiated adenocarcinoma. Endoreduplication is characteristic for both cell lines. Subsequent loss of chromosomes led finally to hypotetraplaid karyotypes with modal chromosome numbers of 66 68 and 70-72 for HS-24 and SB-3, respectively. The structural analysis was performed by G- and C-banding. Stable overrepresentation of chromosomes 7, 8, 12, and 16 was found. Both cell lines developed a characteristic set of disomic and stable markers. Chromosomes involved in markers were 1, 2, 5, 6, 10, 11, 16, and 17. Consistent numerical and structural normality for chromosomes 4, 18, and 21 was observed.
INTRODUCTION
A n u m b e r of c h r o m o s o m e abnormalities have been found in h u m a n cancers, and the theory of n o n r a n d o m n e s s of certain chromosomal changes in h u m a n malignancies has e v o l v e d progressively from these observations [1], cytogenetic results from leukemic tumors still are more prevalent than those from solid tumors [2]. More solid tumors of the c o m m o n types of cancer, like lung cancer, have yet to be karyotypically c h a r a c t e r i z e d [3]. Certain distinctive cytogenetic abnormalities have been reported to occur in lung cancers: a del(3)(p14p23) in small cell carcinomas and occasionally in nonsmall cell carcinomas [4-9] and structural or numerical changes i n v o l v i n g chromosome 7 in non-small cell carcinomas [10], and c h r o m o s o m e 8 and 9 in adenocarcinomas [11, 12]. We report the c h r o m o s o m a l analysis of two permanent h u m a n lung cancer cell lines derived from a squamous cell carcinoma (HS-24) and a metastasis of an a d e n o c a r c i n o m a (SB-3), using G- and C-banding techniques. These cell lines might be of further interest as they exhibit proteolytic enzyme activities possibly relevant in invasive processes [13, 14] and expression of the mucose proteinase inhibitor (MPI) [15]. From the Thoraxklinik Heidelberg-Rohrbach, Department of Clinical Chemistry (M. E.. G. T., W. E.), and German Cancer Research Center (W. P., E. S.), Heidelberg, F.R.G. Address reprint requests to: Dr. E. Spiess, Institute of Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, F.R.G. Received October 23, 1989; accepted February 14, 1990.
185 © 1990 Elsevier Science Publishing Co., Inc.
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Cancer Genet Cytogenet 49:185 198 (1990) 0165-4608/90/$03.50
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MATERIALS AND METHODS Tumor Cell Lines The two h u m a n lung cancer cell lines HS-24 and SB-3 were obtained from surgically dissected tumors of male patients from the Thoraxklinik Rohrbach (Heidelberg, F.R.G.). The cell line HS-24 was established in 1984 from a metastasizing squamous cell carcinoma of the right u p p e r pulmonary lobe, which was staged as T:~N2M1 according to the method of Mountain et al. [161. The cell line HS-24 showed evidence of squamous cell carcinoma origin: positive immunostaining with antikeratin antibodies as well as intercellular bridges which correspond with d e s m o s o m e - t o n o f i l a m e n t complexes as d o c u m e n t e d by ultrastructural studies. The cell line SB-3 was established in 1986 from a poorly differentiated adenocarcinoma in the area of the adrenal glands which was diagnosed as metastasis of a submucous, poorly differentiated adenocarcinoma of the left u p p e r pulmonary lobe. The SB-3 cell line showed evidence of adenocarcinoma origin: positive immunostaining for keratin and reduced homotypic adhesion. A protein preferentially synthesized by lung cells, the mucose proteinase inhibitor (MPI), was detected in the two cell lines according to Western blot analysis [15]. Both cell lines have been cultivated for more than 40 passages, showing obvious immortality and the transformed p h e n o t y p e in vitro. The HS-24 cell line was tumorigenic w h e n t r a n s p l a n t e d subcutaneously in nude mice. SB-3 did not show this ability. Clones of the HS-24 cell line were obtained by limiting dilution and soft agar cloning.
Cell Culture Cells were grown in RPMI 1640 m e d i u m s u p p l e m e n t e d with the following components: NaHCO:~ 24 raM, 10% fetal calf serum, l,-glutamine 4 mM, penicillin 100 IU/ ml, and s t r e p t o m y c i n 100/~g/ml. M e d i u m and all s u p p l e m e n t s were from Biochrom (Berlin, F.R.G.), tissue culture flasks were from Corning (Coming, NY), and trypsin (1 : 250) was from Difco (Detroit, MI). After reaching confluency {5 x 10 ~ and 2 × 10 ~ cells in HS-24 and SB-3 cultures, respectively) cultures were split 1 : 5 and 1 : 2 for HS-24 and SB-3, respectively, and incubated at 37°C in a water-saturated atmosphere (95% air and 5% CO2).
Cytogenetic Analyses Metaphase cells were obtained by blocking the cells in the exponential growth phase of the culture with Colcemid (1-4/zg/ml, Demecolcin; Serva: Heidelberg, E.R.G.) for 1 5 - 3 0 minutes. The cells were harvested by trypsin-EDTA (TED) treatment and r e s u s p e n d e d in a h y p o t o n i c solution of 75 mM KCI. After incubation at 37°C for 20 minutes, cells were fixed by three changes of m e t h a n o l : g l a c i a l acetic acid (3:1; Carnoy) at 4°C for 30 minutes and spread by d r o p p i n g on cold wet glass slides. For c h r o m o s o m e identification and localization of chromosome changes, G-banding was performed by a modification of the G-band technique of Seabright [17]. After storage for 2 - 3 weeks, the slides were treated with 0.025-0.1% trypsin (Boehringer; Mannheim, F.R.G.) in phosphate-buffered saline (PBS) for 20 60 seconds, rinsed briefly in PBS, anc~ treated with 3% Gurr Giemsa {BDH Chemicals, Poole, England) in SOrensen buffer (pH 6.8) for 10-12 minutes. Staining of centromeric heterochromatin (C-bands) was performed essentially according to the method of S u m n e r [18]. Preparations were examined with a Zeiss photomicroscope II. At least 96 u n b a n d e d metaphases were evaluated for numerical chromosome determinations for each cell
H u m a n Lung Carcinoma Cell Lines
18 7
line and passage. During this process, we tried not to avoid less-than-perfect metaphases so that abnormal cells would not be eliminated because of subjectivity. For structural evaluations, 16 and 15 G-banded metaphase spreads with the chromosome n u m b e r s close to the modal n u m b e r were photographed and karyotyped for the cell lines HS-24 and SB-3, respectively. Karyograms of each cell were arranged according to the International System for H u m a n Cytogenetic Nomenclature [19]. RESULTS Cell Line HS-24
Three h u n d r e d sixteen metaphase spreads were evaluated from passages 13 and 37. During this propagation period, no obvious alteration in the modal chromosome n u m b e r was observed. Figure 1 shows the chromosome distribution in this population; metaphases with less than 40 and more than 100 chromosomes were not considered. A subclone with a near-diploid karyotype appeared to be developing; this m i n o r population was not further analyzed. The tumor cell line HS-24 is characterized by a hypotetraploid karyotype with a modal chromosome n u m b e r of 66-68. About 60% of all metaphases had 63-71 chromosomes: 24% of all metaphases showed a modal chromosome n u m b e r between 66 and 68. We also analyzed several clones derived from the HS-24 population: all of them had the same numerical karyotype as the original population. Cells isolated from HS-24 tumors in nude mice also had similar numerical characteristics. Analysis of chromosomes after G-banding was performed with metaphase plates showing 60-72 chromosomes. Sixteen plates, eight from passage 15 and eight from passage 34, were analyzed. A representative spread is shown in Figure 2. Statistical evaluation showed a chromosome n u m b e r of 66 67 with 36 37 normal and 30 abnormal chromosomes. In the group of the abnormal chromosomes, we identified 16 marker chromosomes. Minute and double minute (drain) chromosome structures were not found.
Numerical changes. In modal terms, chromosomes 1, 5, 6, 15, 19, and the Y-chromosome were nullisomic; chromosomes 9, 10, 13, 22, and the X-chromosome were monosonlic: (:hronlosonles 2, 4, 14, 17, 18, 20, and 21 were (lisomic: chromosome 16 was between di- and trisomic; chromosomes 3.7, 11, and 12 were trisomic: and chromosome 8 was tetrasomic (Fig. 3). Differences between the two passages were found in only 10 chromosomes (Table 1): chromosomes 10, 11, 16, and the X-chromosome showed a loss during cell propagation, whereas chromosomes 2, 4, 8, 13, 20, and the Y-chromosome showed gain between passages 25 and 34: e.g., trisomy 16 in passage 15 was lost as it did not appear in passage 34; but # 8 appeared in four copies in passage 34.
Structural changes. Identification of the markers is shown in detail in Table 2. Of the 16 identifiable marker chromosomes, only mar2, mar7, marl0, marl2, and m a r l 4 were disomic (Fig. 4). Mar5, mar9, and m a r l 6 disappeared between passages 15 and 34, whereas mar6 emerged in this period (Table 2). C-banding revealed two dicentric chromosomes. Cell Line SB-3
Two h u n d r e d sixty-three metaphase plates taken from passages 24 and 36 were evaluated numerically. Between these passages only minor numerical differences existed (Fig. 5); therefore, both evaluations were combined. A modal chromosome
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M. Erdel et al.
HS-24 16
P15 (N=204)
14 .
6O
12
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o8
2 o
I
4O
5O
6O
7O
n'
80
1
'
I
90
100
t6 14 O0 qP 69
P.37 (N=112)
12
0 10 _cz O_
08
~4 2 0
1
4O
50
60
'
,•i] 70
80
'
90
I
100
number of chromosomes/metaphase F i g u r e 1 Distribution of chromosome numbers in 316 metaphase plates of the cell line HS24; 204 metaphases w e r e from passage 13, and 112 were, from passage 37. Bars represent a class width of one chromosome.
n u m b e r of 7 0 - 7 2 was thus found, i n d i c a t i n g a h y p o t e t r a p l o i d k a r y o t y p e {Fig. 51. M e t a p h a s e s w i t h less than 40 or m o r e than 100 c h r o m o s o m e s w e r e not c o n s i d e r e d . A b o u t 40% of all m e t a p h a s e s s h o w e d c h r o m o s o m e i m m b e r s b e t w e e n 67 and 75. Nearly 20% of all m e t a p h a s e s s h o w e d the m o d a l c h r o m o s o m e n u m b e r of 7 0 - 7 2 . Structural analysis was p e r f o r m e d with eight and seven m e t a p h a s e plates from passage 24 and 36, r e s p e c t i v e l y . Figure 6 s h o w s a r e p r e s e n t a t i v e karyogram. The analysis s h o w e d 53 55 normal c h r o m o s o m e s and 16 a b n o r m a l c h r o m o s o m e s (total, 69-71}. M i n u t e or drain c h r o m o s o m a l structures w e r e not detected.
189 _M2 M3f~
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Figure 2 G-banded karyotype of a representative HS-24 metaphase plate; marl to marl6 is designated M1 to M16.
F i g u r e 3 Over- and underrepresentation of individual chromosomes m 16 metaphase plates of G-banded karyotypes of HS-24 cells. Bars show the deviation of the respective chromosomes from disomy (2n). Vertical lines show range of chromosome numbers found for each metaphase.
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chromosome
190
M. Erdel et al.
Table 1
O c c u r r e n c e of n o r m a l c h r o m o s o m e s in HS-24 at p a s s a g e s 15 a n d 34 No. of c h r { } I n o s o m e s 'j
Chromosome
Pl 5 modal
P34 modal
Change between passages None
1, 5, 6, 15, 19
0
{}
9, 22
1
1
None
14, 17, 18, 21 3, 7, 12
2 3
2 3
None N{me
10
1
{}
Loss
11 16 X 2, 20 4 8
4 3 2 1 2 2 3
3 2 1 2 3 4
Loss Loss L{}ss Gain Gain Gain
0 {}
1 0-1
Gain
13 Y
Gain
" Data from eight metaphases h}r each passage.
Table 2
M a r k e r c h r o i n o s o m e s of HS-24: I d e n t i f i c a t i o n a n d o c c u r r e n c e at p a s s a g e 15 a n d 34 {:ells with marker
Marker
Identification
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
del(1){q24} psu dic{15)t(15;?;1)(p11;?:p127] der(1]t(1;?}{p12:~) der(5}t(5;10)(pl l;ql 1) der{5)t(5;?}(p 11 :?) der(5)t(5;?)(pl I :?) dup(9)(q12--,qter) t{13q14q) der(16}t(13;16}{q12;q24) 17p+ ? der{15}t{15;?}{p12;?} inv ins{6}{q12pterp12) del{mar12)(q22} dic i{5p} i{12p}? der(2)t(1;2}(pl 3:q24)
P15
P34
50
100
100
100
100 87.5 75
B7.5 100
100 62.5 62.5 100 87.5 100 87.5 1{}0 12.5 66.7
75 75 37.5 100 75 1{}{} 12,5 11}0 50
>2 C{}pies
+
+
t + +
" Data from eight nletaphases for each passage.
Numerical changes. T h e r e s u l t s are s h o w n in Figure 7. T h e r e w a s o n l y o n e n u l l i s o m y , c h r o m o s o m e 11, a n d o n e m o n o s o m y , c h r o m o s o m e 1; d i s o m y w a s f o u n d for c h r o m o s o m e s 3, 4, 6, 7, 9, 10, 13, 14, 18, 19 a n d 21; t r i s o m i e s w e r e f o u n d for c h r o m o s o m e s 2, 15, 16, 20, a n d 22; b e t w e e n 3 a n d 4 c o p i e s w e r e f o u n d for c h r o m o s o m e 5, a n d t e t r a s o m i e s w e r e f o u n d for c h r o m o s o m e s 12 a n d 17. O n l y m i n o r d i f f e r e n c e s b e t w e e n p a s s a g e s 24 a n d 36 b e c a m e o b v i o u s (Table 3): o n e c o p y of c h r o m o s o m e 5 w a s lost d u r i n g t h e p r o p a g a t i o n , w h e r e a s a gain b e t w e e n p a s s a g e s 24 a n d 36 w a s f o u n d in six
191
H u m a n Lung Carcinoma Cell Lines
15
C
a M3
M2
M1
M4
M6
M5 6
C
C D
U ~i!¸i I~
M7
M13
M14
M8
M15
U9
MIO
Mll
M12
M16
Figure 4 Identified marker chromosomes of HS-24. Permanent and diploid markers are boxed. The respective mother chromosomes are indicated by their nmnber: (c) C-banded chromosomes. Marl to marl6 is designated M1 to M16.
chromosomes, i.e., a tetrasomy of chromosomes 7 and 12 and trisomy of chromosomes 3, 4, 8, and 13 that existed only in the later passage.
Structural changes. Among the group of abnormal chromosomes, we could identify six reliable marker chromosomes ( m a r l - m a r 6 ) (Table 4, Fig. 8). Identification of the marker chromosomes is summarized in Table 4. Marl, mar2, mar4, and mar5 appeared with constant disomy in both passages in 100% of the cells. Mar3 and mar6 were monosomic and appeared in passage 24 in nearly 100% of the cells but were reduced later to 86% (mar3) and 43% (mar6) in passage 36. C-banding showed that all chromosomes were monocentric. DISCUSSION
The two tumor cell lines HS-24 and SB-3, established from h u m a n lung tumor material, were karyotyped. Both cell lines exhibited a hypotetraploid karyotype with modal chromosome n u m b e r s of 6 6 - 6 9 and 70-72 for HS-24 and SB-3, respectively. Such modal n u m b e r s and the broad range of chromosome distribution are frequently found in tumors and tumor-derived cell lines [12, 20-22]. A comparison between the two cell lines shows that their karyotypes are of different character. HS-24 is characterized by a high n u m b e r of nullisomies and limited trisomies and tetrasomies, but it showed a high n u m b e r of structural aberrations, i n c l u d i n g 16 persistent marker
192
M. Erdel et al.
SB-3 16 o
P24 (N=167)
14 co 12
._C~10 "
•6 ~4 2
nnnlm,O
o
40
50
' 60
70
80
I
I
90
100
16 -
14
P36 (N=96)
03 12 @ 03
o 10
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1
I
40
5O
6O
'
I4 '
7O
I
8O
'
1
I
90
IO0
number of chromosomes//metophose Distribution of chromosome,numbers in 263 metaphaseplates of SB-3 cells: comparison between passages 24 (167 metaphases) and 36 (96 metaphases). Bars represent a class width of one chromosome.
Figure 5
chromosomes. SB-3 had one nullisomy but large numbers of trisomies and tetrasomies and 6 marker chromosomes. Multiple endoreduplications and subsequent loss of chromosomes have been observed in various h u m a n tumor cell lines in culture [22, 23]. For about 25 passages, the modal n u m b e r s of SB-3 and HS-24 populations were relatively stable. We could not determine the time of the tetraploidization for either culture, but it appeared to be a singular event that occurred either during tumor progression in vivo or in the beginning phase of the in vitro culture. Nevertheless, minor changes in n u m b e r of normal chromosomes (Tables 1 and 3) and marker chromosomes (Tables 2 and 4) did occur during in vitro culture, e.g., gain (mar6) and losses (mar5, mar9, marl6) in
193 M1
1
2
M2 /~
4
3
M3
M4
6
i=
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8
9
7
5 M5 ,,
~¢tlm 10
11
12
16
17
18
21
22
M6 dt~
13
14
19
20
15
3(1?
Qt
X
Y
8
7% ~ % unidentified
marker:
I
?
13
4?
5p?
F i g u r e 6 G-banded karyotype of a representative SB-3 metaphase plate• Marl to mar6 is designated MI to M6.
Figure 7 Over- and underrepresentation of individual chromosomes in 15 metaphase plates of G-banded karyotypes of SB-3 cells. Bars show deviation of the respective chromosomes from disomy (2n). Vertical lines show range of chromosome numbers found for each metaphase,
~
2
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1 2 3 4 5 6 7 8 g 10 1112131415161"71819202122X
chromosome
2n
y
194 Table 3
O c c u r r e n c e of n o r m a l c h r o m o s o i n e s in SB-3 at p a s s a g e s 24 a n d 36 No. o f c h r o i i l o s o m e s e~
Chromosome
P24 modal
P36 modal
Change between passages
11 1, X, Y 6, 9, 10, 14, 18, 19, 21 2, 15, lb, 20, 22 17 5 3, 4, g, 13 7 12
0 1 2 3 4 4 2 2 3
0 1 2 3
None None None None None Loss (;ain Gain Gain
3 4 3 3 4 4
" Data from eight metaphases for P24 and seven metaphases tor P36.
Table 4
Marker chromosomes 24 a n d 36
of SB-3: I d e n t i f i c a t i o n a n d o c c u r r e n c e at p a s s a g e s
Cells with marker (°/o)" Marker
Identification
P24
P36
>2 Copies
1 2 3 4 5 6
inv dup(1)(q32--,q24) der(6)t(6:11)(q26;ql 3) 7q+ 10q+ del{11){q21) der(7)t(7;14}(q11;q11)
100 100 90 lfl0 10{} 100
100
+
100
+
86 100 100 43
+ +