(CANCER RESEARCH 49. 3355-3361, June 15, 1989]
Karyotypic Evolution of a Human Undifferentiated Large Cell Carcinoma of the
Lung in Tissue Culture1
Dennis R. Burholt,2 Stanley E. Shackney, Deborah M. Ketterer, Agnese A. Pollice, Charles A. Smith,
Kathryn A. Brown, HarÃ-anR. Giles, and Brian S. Schepart
Cancer Cell Biology and Genetics Laboratory, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania 15212 fD. R. B., S. E. S., A. A. P., C. A. S., K. A. B.J;
Department of Obstetrics/Gynecology, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212 [D. M. K., H. R. G.J; Department ofMicrobiology /Immunology, The
Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129 [B. S. S.]
ABSTRACT
Serial cytogenetic studies were performed on a cell line derived from
a pleural effusion from a patient with undifferentiated large cell carcinoma
of the lung. The initial sample had a broad range of chromosome numbers
per cell, with a hypodiploid/pseudodiploid stem line and a hypotetraploid
sideline. A sequence consisting of a doubling of chromosome number per
cell followed by chromosome loss was observed repeatedly during 40
culture passages. The presence of metaphase spreads showing evidence
of endoreduplication suggested this as a likely mechanism for the dou
bling of chromosome number per cell. Eleven marker chromosomes were
observed in the cells of the primary sample; these markers persisted
through all subsequent passages. Chromosomes 1, 2, 6, 7, 8, 11, and 16
were consistently overrepresented; each of these chromosomes was in
volved in marker formation. Chromosomes 4, 5, 9, 10, 19, 21, and 22
were consistently underrepresented. Every chromosome, either in its
normal form and/or as part of a marker, was represented on the average
by at least one copy per diploid cell. Eighteen new marker chromosomes
were observed during the course of cell cultivation; one of these evolved
into a clonal marker over the course of six cell passages. Of the new
marker chromosomes that were formed during the observation period,
the majority were found in hypotetraploid cells.
INTRODUCTION
In cytogenetic studies of human lung cancers multiple nu
merical and structural chromosomal abnormalities are com
monly observed (1-10). The frequent finding of aneuploidy by
flow cytometry in human lung cancers (11-13) also reflects the
high frequency of cell subpopulations with numerical chromo
somal abnormalities in these tumors. Although certain distinc
tive cytogenetic abnormalities have been reported to occur in
human lung cancers [a deletion involving 3p occurring fre
quently in small cell carcinoma (10) and occasionally in nonsmall cell carcinoma (7), and structural or numerical chromo
somal abnormalities involving chromosome 7 in non-small cell
cancer (14)], a striking feature of these tumors is their cytoge
netic heterogeneity, both among different tumors and among
different cells within the same tumor.
In this paper we report the results of sequential cytogenetic
studies in a human undifferentiated large cell lung cancer cell
line that was established in our laboratory. Although this cell
line exhibited extensive heterogeneity with respect to both
numerical and structural chromosomal abnormalities, the data
suggest that the genetic evolution of this cell line followed an
orderly and predictable pattern. A sequence that consisted of a
doubling of chromosome number per cell followed by chromo
some loss occurred repeatedly, accounting for most if not all of
the observed variability in chromosome numbers per cell over
time. Despite large cell to cell variations in chromosome num-
ber, certain chromosomes were consistently overrepresented
and others were consistently underrepresented. Each of the
overrepresented chromosomes was associated with a distinctive
structural abnormality. These findings are presented below in
detail.
MATERIALS
AND METHODS
Cell line EuRo was derived from a malignant pleural effusion from
a male patient with an undifferentiated large cell carcinoma of the lung.
The patient had received prior radiotherapy and chemotherapy with
cisplatin and etoposide 4 weeks before the initiation of the cell line.
The cells were grown in Corning 25-cm2 polystyrene tissue culture
flasks (Corning, NY) in 5 ml of RPMI 1640 with L-glutamine and 4(2-hydroxyethyl)-l-piperazineethanesulfonicacid
buffer. Twenty% heat
inactivated fetal calf serum, 0.3% gentamicin, 8 mg/liter insulin, and 8
mg/liter transferrin were added to the medium. The cell cultures were
maintained at 37°Cin a humidified 5% CO2 atmosphere. Cells were
seeded at an initial concentration of 1 x 10' cells/ml. The cells grew
suspended in the culture medium. Smears of these cells exhibited
cytological features that were diagnostic of an undifferentiated large
cell carcinoma. Cell cultures were examined microscopically 2 to 3
times/week and were passaged when the medium became depleted,
usually at a cell concentration in the range of 7 x 105-1 x IO6 cells/
ml. Except for the first several transplant passages, which were carried
out at 3- to 4-week intervals, cells were transplanted at weekly intervals
for a total of 40 passages. Cells from passage 40 were frozen and stored
at -80°C.
Metaphase cells were obtained during the course of serial passages
by blocking with 0.2 iig/ml Colcemid overnight. The cells were har
vested, treated with 0.075 M KC1 at 37°Cfor 8 min, and then fixed in
methanol:glacial acetic acid (3:1). Cell suspensions were dropped on
wet slides at room temperature. The slides were dried overnight at
60°C.GTG banding was performed using a modified Klinger technique
(15). Metaphase spreads were evaluated with the aid of a karyotype
image editor (Katie; Greenwood Scientific Instruments, Greenwood,
SC). The karyotype image editor system generates a video image of the
karyotype spread and digitizes, enhances, and formats the image elec
tronically. Prints were obtained by using a Lasertechnics 300D high
resolution printer (General Imaging Corporation, Gainesville, FL).
Karyotypes of each cell were arranged according to the International
System for Human Cytogenetic Nomenclature (16).
RESULTS
Numerical Chromosomal Changes during Serial Culture. Met
aphase cells obtained directly from the malignant pleural effu
sion of patient EuRo had a broad range of chromosome num
bers, spanning 40 to 96 chromosomes/cell (Fig. IA). The
stemline contained between 40 and 46 chromosomes/cell
(modal chromosome number, 44) and comprised 57% of the
Reeeived 8/8/88; revised 3/6/89; accepted 3/22/89.
total mitotic cells observed. A sideline, comprising approxi
The costs of publication of this article were defrayed in part by the payment
mately 15% of the total, had between 74 and 82 chromosomes/
of page charges. This article must therefore be hereby marked advertisement in
cell. Cells were also present with chromosome counts between
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' Supported by Allegheny-Singer Research Institute.
the two modal peaks, as well as chromosome counts above the
2To whom requests for reprints should be addressed, at Cancer Cell Biology
secondary peak.
and Genetics Laboratory, Allegheny-Singer Research Institute, 320 E. North
During the early culture passages, the stem line remained in
Avenue, Pittsburgh, PA 15212.
3355
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KARYOTYPIC EVOLUTION OF A HUMAN
Fig. 1. Distribution of chromosome num
bers per cell, expressed as a percentage of the
total number of metaphases observed, for the
primary sample (A), and for passages 1 (B), 4
(C), 8 (D), 14 (£),26 (f), 28 (G), 30 (//), and
31 (/). The number of metaphase cells ob
served for each passage is indicated.
,
diploid and tetraploid positions. Chromosome
numbers per cell are grouped four to a histo
gram class. Thus, for example, the histogram
bar centered on 46 chromosomes per cell in
cludes cells with 44 to 47 chromosomes.
LUNG CANCER
0
Ü
0)
0.
92
69
>115
46
69
92
>115
46
69
92
>115
Chromosome Number Per Cell
the diploid to hypodiploid range (Fig. 1, B-D). However, the
chromosome number per cell of the sideline decreased to be
tween 56 and 64 at passage 1 (Fig. IB) and decreased further
to between 49 and 53 at passage 4 (Fig. 1C). At passage 8, a
hypotetraploid sideline reappeared (Fig. ID). With further cul
tivation, the original hypodiploid stem line was greatly reduced,
comprising less than 5% of the cells observed by passage 14
(Fig. IE); the stern line exhibited hypotetraploidy, with chro
mosome numbers ranging predominantly between 80 and 86.
This pattern remained stable through passage 26 (Fig. IF). At
passage 28, a downward shift in the chromosome numbers of
this hypotetraploid stem line was observed (Fig. IG) and by
passage 30, the modal chromosome number per cell fell to the
hyperdiploid range (Fig. IH). At passage 31, a distinct hypo
tetraploid stem line was reestablished, with a modal chromo
some number of 76 (Fig. 17).
Endoreduplication was observed in four metaphase spreads
of the 661 metaphase spreads counted (Fig. 2), suggesting that
the hypotetraploid sidelines may have arisen by a process of
doubling of chromosome number with subsequent chromosome
loss. This is supported further by the observation that hypo
tetraploidy developed rapidly (e.g., Fig. l, H and 7) and by the
commonality of marker chromosomes and other karyotypic
features in the near-diploid and hypotetraploid cells (see below).
Karyotype Analysis. A total of 51 cells were karyotyped: nine
cells from the primary effusion; three from passage 3; three
from passage 4; two from passage 8; eleven from passage 13;
six from passage 26; five from passage 31; three from passage
32; two from passage 34; and seven from passage 38. The
chromosomes from cells of the primary sample and the early
passages were partially banded and were more difficult to
karyotype than those from later passages. There were no normal
diploid karyotypes among the 51 cells analyzed. All of the cells
from the near-diploid stem line that was present in early pas
sages exhibited extensive numerical and structural chromo
somal abnormalities. Shown in Fig. 3 is a hypodiploid cell from
the primary sample which contained multiple copies of chro
mosomes 2, 8, and 20 and only one normal copy of chromo
somes 1, 4, 7, 9, 15, 16, 17, and 22. There were no normal
copies of chromosomes 10, 11, 14, 19, 21, and Y. Nine marker
chromosomes were present in this cell.
A total of 11 stable marker chromosomes and a group of
double minutes were defined in cells from passages 13 through
38. The presence of all of these marker chromosomes was then
confirmed in the primary sample and in early culture passages.
All 11 stable marker chromosomes are present in the karyotype
shown in Fig. 4; 9 of these 11 markers correspond with those
present in the hypodiploid cell shown in Fig. 3.
The marker chromosomes were defined as follows:
Ml:
M2:
M3:
M4:
M5
M6:
t(l;?;?) (p22). The short arm is probably a com
plex, undefined translocation.
t(l;?) (p22). Possibly a terminal deletion of both
the short and long (Ip36) arms of Ml.
i(7p)
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KARYOTYPIC EVOLUTION OF A HUMAN
LUNG CANCER
Fig. 2. Metaphase spread from passage 3
showing evidence of endoreduplication.
M7:
M8:
M9:
MIO:
Mil:
t(14;?)(q32)
t(ll;16)(ql3;q23)
t(16;?)(q24)
inv(16)(q21q24)
t(8;19)(q23;ql3)
Although none of the 51 cells had identical karyotypes,
certain similarities were observed among cells within a given
passage and among cells of different passages. There were no
normal copies of chromosomes 11, 14, and 19 in any of the
cells. However, each of these chromosomes was represented in
at least one marker. Chromosome 11 was represented in mark
ers M4, M6, and M8. Chromosome 14 was represented in
marker M7. Chromosome 19 was represented in marker Mil.
Normal chromosomes 2, 3, 8, and 20 were overrepresented
both in near diploid and in near tetraploid cells. Other normal
chromosomes, particularly 4, 5, 9, 10, 21, and 22 were underrepresented. The 11 marker chromosomes and the double min
utes were present throughout all of the cell passages. The same
marker chromosomes were present in both the near-diploid and
hypotetraploid cells. These markers were often represented by
multiple copies per cell, particularly in cells with large numbers
of chromosomes. The commonality of markers in the neardiploid and hypotetraploid cells supports the mechanism of
repeated doubling of chromosome number per cell followed by
chromosome loss.
In order to obtain quantitative estimates of the frequencies
of individual chromosomes per cell in the presence of chromo
some doubling and chromosome loss, chromosome frequencies
per cell were normalized with respect to total chromosome
number per cell, using the diploid number of 46 chromosomes
per cell as a reference standard. Thus, for example, if there
were 4 copies of a given chromosome in cells with 92 chromo
somes, the normalization procedure would yield 2 chromosome
copies per diploid cell. Similarly, if there were 3 copies of each
chromosome in a cell with a total of 69 chromosomes, there
would still be 2 copies per diploid cell. The normalized chro
mosome frequencies shown in Fig. 5 are expressed in terms of
the deviation from the normal number of chromosomes per
diploid cell (2 of each autosome per diploid cell and 1 of each
sex chromosome) plotted in relation to tissue culture passage
number. When a normal chromosome was represented in whole
or in part in a marker chromosome, this was included in the
calculation of normalized chromosome frequency. In cases
where all or part of a p arm and/or q arm of a normal
chromosome was involved in marker formation, the represen
tation of each chromosome arm was expressed separately.
It is apparent from Fig. 5 that all of the normal chromosomes
were represented on the average by at least one copy per diploid
cell. Several chromosomes were consistently overrepresented in
the karyotypes throughout the period of cell culture, and the
number of extra copies per cell did not change significantly
over time. Without exception, every chromosome that was
overrepresented was involved in a structural chromosomal ab
normality. Chromosome 1 was present in its normal form and
it was also partially represented in markers Ml, M2, and M3.
This resulted in the overrepresentation of parts of the short
arm and all of the long arm of chromosome 1. There were, on
the average, 0.5 to 1 extra copy of normal chromosome 2 per
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1t' i m x
KARYOTYPIC EVOLUTION OF A HUMAN
i
M1M2
(
M3
m i
U
8
•¿A
I
13
LUNG CANCER
M7
I
15
II
H8 M916
M6
2
M10
IXX
20
22
If
XI
12
A
17
18
I
DM
Fig. 3. G-banded karyotype of a cell from the primary sample consisting of 31 normal chromosomes, 9 of the 11 identified marker chromosomes, and a double
minute.
diploid cell. The involvement of the short arm of chromosome
2 in marker M3 resulted in 2 to 3 extra copies per diploid cell.
The long arm of chromosome 6 was present as part of M4,
resulting in the overrepresentation of that portion of the chro
mosome. Although normal chromosome 7 was generally underrepresented in the karyotypes, the short arm of chromosome
7 was involved in the formation of isochromosome M5, pro
ducing two additional copies of 7p per diploid cell. Chromo
some 8 was overrepresented both as a normal chromosome and
as part of marker Mil. The long arm of chromosome 11 was
involved in the formation of three markers, resulting in its
overrepresentation. A portion of the short arm of chromosome
11 was involved in only one of these markers. The terminal
portion of 1Ip was not detected in any of the cells. Chromosome
16 was the most highly overrepresented of all the chromosomes.
It was present in its normal form, and it was also involved in
the formation of markers M8, M9, and MIO.
It is apparent from Fig. 5 that several chromosomes were
consistently underrepresented in all passages. Chromosomes 4,
5, 9, 10, 19, 21, and 22 were represented, on the average, by
only one copy per diploid cell throughout the period of study.
Chromosomes 17 and 18 were underrepresented in the early
passages but tended toward normal representation in the later
passages.
When the 11 original marker chromosomes were examined
individually, it was found that each was represented on the
average by one copy per diploid cell, with the exception of
marker M3. Marker M3 was represented by one copy per
diploid cell in the initial tumor sample; by passage 3, the average
number of copies of M3 rose to two copies per diploid cell and
remained at this level through all subsequent cell passages.
The data presented in Fig. 5 represent average values of
chromosome frequency per cell, based on all of the cells karyotyped in a given passage. Although, on the average, each
chromosome was represented at least once per diploid cell, it
was often the case that both copies of one or more normal
chromosomes were missing in individual cells, and that this
chromosomal material was not recognizable in any of the
markers in these cells. Of the 51 karyotypes analyzed, 30 were
missing both copies of at least one normal or marker chromo
some. There was no single chromosome that was lost prefer
entially, suggesting that chromosome loss was largely a random
process. All but one of the cells that contained at least one copy
of every chromosome had chromosome counts that were above
the triploid range. In contrast, cells that were devoid of three
or more normal chromosomes were almost all hypodiploid.
Most of these hypodiploid cells were from the early passage
stem line that did not survive (see Fig. 1), suggesting that cells
devoid of multiple normal chromosomes either were nonviable
or were unable to compete favorably with cells that retained a
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KARYOTYPIC EVOLUTION OF A HUMAN
LUNG CANCER
I
f|(*
s»*
1
Ml
M2
M3
lì
Ml«
u mitii iso«
m
7
13
MS
9
8
10
M6
12
S$¿MARK*
ft*¿
«s
M7
M8
16
M9
MIO
18
17
è
Mil
28
21
22
v
X
A ».
Hl 2
DM
Fig. 4. G-banded karyotype of a cell from passage 38 of the culture consisting of 56 normal chromosomes, all 11 of the original marker chromosomes, 2 double
minutes (DM), and a new clonal marker chromosome (M 12).
Fig. 5. Deviations from the diploid chro
mosome copy number per cell for each autosome and the two sex chromosomes in relation
to culture passage number. Data were normal
ized with respect to total chromosome number
per cell and expressed as the deviation from
the expected number of chromosome copies
present in a normal diploid cell. A zero value
indicates that a chromosome or chromosome
arm was present twice per diploid cell. A value
of +1 or +2 indicates that one or two extra
chromosome copies were present per diploid
cell, and a value of —¿
1 or —¿2
indicates that one
or two chromosome copies were missing per
diploid cell. For additional discussion, see text.
V, p arm; O, q arm; •¿,
whole chromosome.
12
18
+1
0
-1
-219
+2 +1
0
-1
-240
20
40
20
40
20
40
20
40
20
Passage Number
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40
KARYOTYPIC EVOLUTION OF A HUMAN
LUNG CANCER
mors. It has been observed during cell transformation in vitro
following SV40 infection (21) and in conjunction with the
spontaneous transformation of mouse fibroblasts (22-24). The
doubling of chromosome number per cell has been found to be
associated with an increase in the growth rate of human tumors
HI vitro (25) and has been found to accompany tumor progres
sion in vivo (26).
The data shown in Fig. 6 raise the possibility that the dou
bling of chromosome number may be associated with an in
crease in the rate of development of new structural abnormali
ties. Shapiro and Shapiro (25) have made similar observations
during the course of serial studies of human gliomas in tissue
culture. In another non-small cell lung carcinoma cell line
established in our laboratory, over 80% of new structural chro
mosomal abnormalities developed in hypertetraploid cells and
fewer than 20% developed in hyperdiploid cells (27). While
these findings suggest a possible relationship between numerical
chromosomal abnormalities and the development of structural
chromosomal abnormalities, further studies will be required to
clarify the nature and the extent of this relationship.
It is apparent from the normalized chromosome frequency
data shown in Fig. 5 that certain chromosomes (1, 2, 6, 7, 8,
11, and 16) were consistently overrepresented in whole or in
DISCUSSION
part, while other chromosomes in the same cells were consist
ently underrepresented, despite large fluctuations in total chro
Karyotype studies in many solid tumors, and particularly in
mosome numbers per cell during the period of study. Although
non-small cell lung carcinomas (1-9), have demonstrated ex
no 2 karyotypes were identical among the 51 that were analyzed,
tensive heterogeneity in chromosome number per cell, as well
the average number of excess copies among the overrepresented
as multiple, complex structural chromosomal rearrangements.
chromosomes was remarkably stable throughout the period of
These were also observed in the present study. The overrepresentation of chromosome 7 has been reported in non-small cell cultivation.
The establishment and perpetuation of a new clonal structural
lung cancer cell lines (14) and other solid tumors (17-20) and
chromosomal abnormality can be taken as prima facie evidence
was also observed in cell line EuRo. The nonrandom chromo
that this structural abnormality has conferred an added growth
somal abnormality del (3) (pi4, p23) that is often found in
advantage
on the cells that contained it, presumably by the
small cell lung carcinoma (10), and occasionally in non-small
activation of a new growth promoting gene or by the inactivacell lung carcinoma (7), was not present in this cell line.
tion of a growth suppressing gene. The first appearance of
Despite the extensive karyotypic heterogeneity in cell line
marker M12 in a single metaphase in passage 31, and its
EuRo, the data suggest that the genetic evolution of this tumor
subsequent detection in three of seven metaphases (43%) at
cell line was an orderly process. A sequence consisting of a
passage 38, indicate the rapidity with which the establishment
doubling of chromosome number per cell followed by progres
and overgrowth of a new marker can occur under in vitro culture
sive chromosome loss in subsequent cell divisions was observed
conditions. Since this new marker did not replace any of the 11
repeatedly during a period of cell cultivation that spanned 40
markers already present, it would be reasonable to assume that
serial passages. These repeated cycles of doubling of chromo
its growth promoting properties must have been at least par
some number with subsequent chromosome loss accounted for
tially additive to those of the established markers.
the heterogeneity in chromosome number per cell.
Marker M3 was represented on the average by two copies
The doubling of chromosome number per cell with subse
per
diploid cell, whereas the other markers were represented by
quent chromosome loss has been noted in many different tuone copy per diploid cell. In order for a chromosome with a
particular growth promoting structural abnormality to become
Q
tu
Qoverrepresented numerically in relation to other chromosomes
with growth promoting properties in the same cell, two condi
£ 1.0tions should be met. (a) One might expect that the growth
E
* 0.8promoting effect would be gene dose dependent; i.e., a cell
n
containing two copies of the chromosome bearing the growth
_i
0 06~
promoting structural abnormality should have a competitive
advantage over a cell that contains only one copy of this
K
0.4 -1
chromosome. (/>)There must be a genetic mechanism in place
co
for preferentially increasing the number of copies of the chro
0.2 -|
mosome that bears the gene dose dependent growth promoting
structural abnormality. Two such mechanisms come to mind.
o-1
The first is repeated nondisjunction of the chromosome bearing
10
20
30
the growth promoting structural abnormality. This mechanism
PASSAGE NUMBER
alone would be expected to produce small, progressive incre
Fig. 6. Modal chromosome number (points) and the frequency of new marker
ments in total chromosome number per cell above the diploid
formation (bars) in relation to passage number. The frequency of new marker
value. The appearance of multiple copies of the Philadelphia
formation is expressed as the number of new marker chromosomes per cells
chromosome prior to and in association with blastic transforkaryotyped in each passage.
3360
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minimum complement of normal genetic material.
New Marker Formation. During the course of growth of the
cells in culture, 18 new marker chromosomes appeared. Of
these new marker chromosomes, 17 were observed in only 1
cell. The remaining new marker chromosome first appeared in
a cell of passage 31 and was then observed in three cells in
passage 38. This new clonal marker chromosome is designated
Ml2 in Fig. 4. It is likely that this chromosome contained a
translocation involving a portion of chromosome 9.
Fig. 6 shows the frequency of new marker formation ex
pressed as new markers per cells karyotyped in each passage.
Also shown in Fig. 6 is the modal chromosome number for
each passage. Because of the technical difficulties in identifying
new markers with certainty in the partially banded chromo
somes of early passages, these passages were excluded from the
analysis. The rate of new marker formation during passages 13
and 26, which spanned a period of relative stability in chro
mosome number per cell, was relatively low in comparison with
the rate of new marker formation during passages 31 and
beyond, when a doubling of chromosome number per cell
occurred.
KARYOTYPIC EVOLUTION OF A HUMAN
LUNG CANCER
mation of chronic myeloid leukemia (28) might conceivably
occur by this mechanism.
Another mechanism for preferentially increasing the number
of copies of one or more chromosomes that bear gene dose
dependent growth promoting structural abnormalities is that of
repeated cycles of doubling of chromosome number per cell
with subsequent random chromosome loss. This would be in
keeping with the data shown in Fig. 1, in which there are
abrupt, large increases in chromosome number per cell over
time, followed by more gradual decreases. Of course, the dou
bling of total chromosome number per cell would produce no
immediate preferential increase in the copy number of any
given chromosome. However, the subsequent random loss of
chromosomes during succeeding cell divisions would generate
progeny with considerable genetic diversity with respect to both
the number and types of chromosomes present in each cell.
This diversity would permit the selection and overgrowth of
those cells with the largest number of chromosomes that bear
activated growth promoting genes the effects of which are
mutually additive, whether the chromosomes bear the same
growth promoting genes or different ones. This would be in
keeping with the data shown in Fig. 5, in which several different
chromosomes with different structural abnormalities were overrepresented in the same cells. The loss of chromosomes has
also been implicated in the expression of recessive phenotypes
in cells that are heterozygous for a recessive alÃ-ele(29).
Although every chromosome was represented at least once
per cell on the average, there were many individual cells in
which both copies of one or more chromosomes were lost.
Almost all of the cells that were missing both copies of three
or more chromosomes were hypodiploid. Almost all of these
cells were found in early passages, as members of a stem line
that did not maintain itself over time (see Fig. 1, .I-/)). It is
clear that these cells could not have been the progenitors of the
hypotetraploid stem line that dominated the later culture pas
sages (Fig. 1, E-I), since all but one of the karyotyped hypote
traploid cells either had at least one copy of all the chromo
somes or had lost both copies of no more than one chromosome.
This would suggest that the complete loss of a substantial
amount of normal chromosomal material either impaired the
viability of individual cells or compromised their ability to
compete successfully over the long term with other cells that
retained a minimum complement of normal chromosomal ma
terial.
The foregoing would suggest still another role for the dou
bling of chromosome number per cell in relation to the genetic
evolution of cell line EuRo. The doubling of chromosome
number per cell provided a reserve pool of normal chromosomes
per cell in order to maintain a minimum complement of normal
genetic material required for cell survival in the face of subse
quent random chromosome loss. Thus, for example, in a pop
ulation of hypotetraploid cells, each of which has at least two
copies of each chromosome, the selection for extra copies of
chromosomes bearing activated growth promoting genes can
proceed with little likelihood that random chromosome loss
will eliminate the last copy of one or more normal chromosomes
that are vital for cell survival. On the other hand, a near-diploid
cell with several overrepresented chromosomes will invariably
have several chromosomes that are underrepresented. In such
a cell, continued random chromosome loss is much more likely
to lead to the total loss of vital normal genetic material that is
on one or more of these underrepresented chromosomes.
cerous effusions. Cancer (Phila.), 36: 1729-1738, 1975.
2. Pickthall, V. J. Detailed cytogenetic study of a metastatic bronchial carci
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and causation of human cancer and leukemia. XX. Banding patterns of
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4. Van der Reit-Fox, M. F., Retief, A. E., and Van Nickerk, W. A. Chromosome
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5. Wake, N., Slocum, H. K., Rustum, Y. M., Matusi, S. I., and Sandberg, A.
A. Chromosomes and causation of human cancer and leukemia. XLIV. A
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3361
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1989 American Association for Cancer Research.
Karyotypic Evolution of a Human Undifferentiated Large Cell
Carcinoma of the Lung in Tissue Culture
Dennis R. Burholt, Stanley E. Shackney, Deborah M. Ketterer, et al.
Cancer Res 1989;49:3355-3361.
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