[CANCER RESEARCH 50, 740-747. February I. 1990]
Nonrandom Chromosome Alterations That Correlate with Progression to
Immortality in Rat TrachéalEpithelial Cells Transformed with
yV-Methyl-yV'-nitro-TV-nitrosoguanidine
Sumiyo Endo, Paul Nettesheim1, Mitsuo Oshimura, and Cheryl Walker2
Laboratory of Pulmonar)- Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 fS. E., P. A/., C W.]; and
Laboratory of Cell Biology, Kanagawa Cancer Center, Research Institute, Nakao-chyo 54-2, Ashahi-ku, Yokohama, Kanagawa-Ken, Japan ¡M.OJ
ABSTRACT
Primary rat trachea! epithelial cells can be transformed in vitro by V
methyl-/V'-nitro-/V-nitrosoguanidine. The earliest recognizable morpho
logical transformant is the enhanced growth variant (EGV), characterized
by enhanced proliferative capacity. Transformed EGV colonies can pro
gress to give rise to immortal cell lines. The purpose of this study was to
determine if specific chromosome changes occur which correlate with
immortalization. A total of 34 EGV colonies were isolated, of which five
were able to progress in culture to become immortal (•!()()population
doublings). Early passages of all fire immortalized cultures exhibited
additional copies of chromosomes 4, 7, and 11 as a common or recurrent
abnormality. These numerical alterations were rarely observed in the
primary EGV colonies from which the cell lines were derived, suggesting
that these alterations occurred during progression. Structural alterations
involving chromosome 1 (resulting in a net gain of Iq) and chromosome
3 (3q) also occurred in four out of five immortalized cultures. In all cases,
structural alterations involving Iq and/or 3q were detected in the primary
EGV colonies from which the immortal cell lines arose. Comparison of
the frequency of the structural and numerical alterations observed in the
immortalized cultures with their frequency in the 29 EGV colonies which
did not become immortal indicated that these changes correlated (/' :
0.005) with the ability to become immortal. These results suggest that
structural alterations occur in primary EGV colonies which predispose
cells to immortalization and that subsequent numerical changes occur
during progression that correlate with acquisition of the immortal phenotype.
proliferation of normal RTE cells to cease, and these cells then
senesce and slough off the dish. In contrast, cells transformed
by chemical carcinogens continue to proliferate after feeder
removal and form morphologically altered foci consisting of
cells with an enhanced growth capacity. Cells isolated from
these colonies can continue to progress to become immortal
and ultimately neoplastic (4).
In an earlier report, we described the ability of the carcinogen,
MNNG, to induce cytogenetic alterations in primary RTE cells
(5). The results of that study indicate that transformed cells
which were aneuploid had a selective growth advantage relative
to diploid cells when the primary transformed foci were replated
on plastic. In that report we were unable to detect specific
chromosome changes that correlated with the morphologically
transformed phenotype, although alterations involving chro
mosomes 3 and 4 were recurrently observed. In a subsequent
series of experiments, however, it became clear that only a
fraction of all primary morphologically transformed colonies
have the potential to become immortal (6). Therefore, if specific
chromosome alterations occur which correlate with the ability
to progress to this next stage in the process of neoplastic
transformation, these alterations would not be recognizable as
"nonrandom" changes at the early morphologically trans
formed stage due to the high proportion of colonies in this
population with finite growth potential. Thus, the purpose of
the present study was to determine whether in those morpho
logically transformed cells with the potential to become im
mortal, any specific chromosome alterations could be detected
that correlated with the immortal phenotype.
INTRODUCTION
The observation in various systems that specific chromosome
aberrations are often associated with neoplasia has led to the
suggestion that such chromosomal alterations may play a role
in the process of neoplastic transformation (1, 2). This is
supported by evidence that many chemical carcinogens can
cause chromosome aberrations (3). However, in the case of
many solid tumors, it remains a key question as to whether the
chromosome alterations observed are in fact causal, or second
ary events which occur during the process of neoplastic pro
gression. It is necessary, therefore, to determine when during
neoplastic transformation specific chromosome alterations oc
cur in order to understand how a given alteration participates
in the carcinogenic process.
We have developed an in vitro model system to study multi
stage chemical transformation of primary rat trachea! epithelial
cells. In this system, primary RTE1 cells are plated on an
MATERIALS
AND METHODS
Transformation of RTE Cells. Primary RTE cells were isolated from
the tracheas of Fisher 344 rats as described previously (7). The RTE
cell suspension was plated on a layer of irradiated mouse 3T3 fibroblast
cells at 2 x 10" cells/60-mm dish in Ham's F-12 medium supplemented
with 5% fetal bovine serum, insulin (1 ng/ml), hydrocortisone (0.4 ^g/
ml), and penicillin (100 units/ml) and streptomycin (100 Mg/ml) and
cultured at 37°Cin a humidified atmosphere of 5% CO2. Methodology
irradiated fibroblast feeder layer, exposed to carcinogen, and
after a recovery period of a few days, the feeder layer is selec
tively removed. Removal of the feeder layer causes the
Received 5/17/89; revised 10/17/89: accepted 10/25/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom reprint requests should be directed.
2 Present address: CUT PO Box 12137. Research Triangle Park. NC 27709.
3The abbreviations used are: RTE. rat trachéalepithelial: MNNG, A'-methylA"-nitro-/V-nilrosoguanidine: EGV, enhanced growth variant: PBS. phosphate
buffered saline.
for transformation of RTE cells has been described in detail elsewhere
(7). Briefly. 24 h after plating the cells were exposed to MNNG (0.3
Mg/ml) for 4 h, refed with complete medium, and 4 days after carcinogen
treatment the feeder layers were removed with EDTA (0.0029;,). The
dishes were then cultured in complete medium for an additional 6
weeks.
Isolation and Culture of Transformed Cells. Individual transformed
colonies were identified by light microscopy and any residual cells
remaining in the dishes or other transformed colonies were removed
from the surface of the dish with a sterile cotton-tipped swab leaving a
single isolated transformed colony per dish. At 8 weeks the dishes were
examined by eye and under the light microscope to ensure that no
residual cells outside the original EGV colony were present in the dish.
One half of each colony was then trypsinized by holding the dish at a
10-45°angle so that only a portion of the colony came in contact with
the trypsin solution. After 2-5 min of trypsin exposure at room tem
perature, the dissociated cells were removed by tritaration with a
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CHROMOSOME
ALTERATIONS CORRELATING
Pasteur pipet, resuspended in 3 ml of complete medium, and plated in
two wells of a six-well dish. The portion of the colony that remained
attached to the dish was refed with complete medium and used for
cytogenetic analysis 24-72 h later.
The transformed cells were then progressively subcultured when they
reached 75% confluence into 25- and 75-cm2 flasks and then split 1:2
into two 75-cm2 flasks (split passage 1). In these experiments all five
WITH PROGRESSION
TO IMMORTALITY
RESULTS
Isolation of EGV Colonies and Immortal EGV Cell Lines
Transformed EGV colonies were generated by exposing pri
mary RTE cells to the carcinogen MNNG. Six weeks after
carcinogen exposure, the transformants could be recognized as
colonies which reached split passage 1 could be subcultured to passage
morphologically altered cells persisting in discrete colonies.
20 (>100 cell doublings). We have recently shown that all transformed
colonies that can be subcultured to split passage 1 continue to replicate
Transformed colonies were scored and isolated by removal of
past 100 cell doublings to acquire indefinite growth capacity (6). Colony
any extraneous cells in the dish with a sterile swab leaving one
forming efficiency was determined between passages 5 and 7. 9 and 11. transformed colony per dish. A total of 34 transformed EGV
and at passage 20 for each cell line by plating cells on plastic tissue
colonies were isolated and 8 weeks after the carcinogen expo
cultures dishes in triplicate at IO2- 5 x IO4cells/60-mm2 dish. Dishes
sure the transformed colonies were passaged to establish cell
were fixed, stained, and scored after 10 days.
lines as shown in Fig. 1. One half of each transformed colony
Statistics. Two statistical analyses were used in the following study.
was removed by trypsinization for the establishment of cell
Determinations of chromosome alterations that correlated with im
lines: the remainder of the colony was prepared for cytogenic
mortality were made using the Fisher exact test (8), which is sufficiently
analysis. The detached cells were seeded into two wells of a sixvigorous for use with small sample sizes (n = 5 for the immortal cell
well dish and were expanded to two 75-cm flasks (passage 1).
lines). In those instances when the observed frequency of a specific
chromosomal alteration in the nonimmortalized population was 0, an
At this stage, cells from one flask were used for cytogenetic
estimate was made of the minimum detectable frequency for a chro
analysis and the cells from the other flask were used to continue
mosome alteration in a primary EGV colony by the following equation:
propagation of the cultures.
Five of the 34 transformed colonies used in these experi
Total number of chromosome alterations observed
ments, EGV-116, 117, 124, 401, and 404 acquired indefinite
Total number of chromosomes/metaphase
growth potential (immortality) and gave rise to cell lines. The
(i.e., 2n = 42xy) x total number of EGV colonies
colony-forming efficiency of all five EGV cultures progressively
Mitotic index data was analyzed using a one-tailed Student's t test. In
increased between passage 1 and passage 20 (Fig. 2). All colo
order to meet the homogeneity of variance assumption for this test, the
nies that survived to passage 1 (Fig. 1) continued to proliferate
analysis was conducted using a logarithmic transformation of the data.
to > passage 20 (>100 cell doublings). Thus, as we have
Cytogenetics. For immortalized cell lines, flasks containing loga
observed previously (6), the ability to reach passage 1 with this
rithmically dividing cells were treated with colcemid (0.005 ^g/ml, final
concentration) in complete medium for l h at 37°C.For primary EGV
regimen can be used as an indicator of the ability of cultures to
colonies, cells in intact colonies were treated with colcemid (0.05 ¿tg/ achieve cell line status. Therefore, experiments were conducted
ml, final concentration) for 2 h at 37°C.After the colcemid treatment,
to determine if specific chromosome alterations which may
the medium was removed and stored in a centrifuge tube. The cells participate in the acquisition of immortality were detectable at
were washed twice with calcium/magnesium-free PBS, which was also
this early stage. Cytogenetic analyses were thus performed at
saved and combined with the original medium. The cells were then
early passages, (passage 1 or 2) on all five cultures which became
treated with PBS containing 0.05% trypsin and 0.02% EDTA for 5 immortal to answer the following questions: (a) are immortal
min (immortalized cell lines) or 10 min (intact colonies) at 37°C.After
ized cell lines karyotypically abnormal?, (b) if so, do they
all the cells on the plate had detached, this solution was combined with
contain specific chromosome alteration(s)?
the aliquots of the original medium and PBS washes. The cells in this
combined solution were then centrifuged (1200 x g, 5 min) and resus
pended in 0.075 M KC1 for 30 min at room temperature. After hypotonic treatment, a fixative consisting of methanol:glacial acetic acid
(3:1, v/v) was added slowly to the cell suspension. The cells were then
pelleted, the supernatant removed, and fresh fixative was added. Centrifugation and resuspension in fresh fixative were repeated at least
three times. The cells were placed onto clean microscope slides on wet
papers and air dried.
Karyotyping. The chromosome slides were banded according to a
quinacrine fluorescence banding method (9). Chromosome analyses
were made according to the standardized nomenclature for rat chro
mosomes (10). At least 30 metaphases were analyzed to determine the
modal karyotypes of the cells of every cell line and primary transformed
colony. Recurrent chromosome abnormalities (observed in at least 20%
of metaphases) and common chromosome abnormalities (observed in
at least 80% of the metaphases) were noted.
For each cell line, and the primary EGV colony from which it was
derived, chromosome analyses consisting of 10 banded karyotypes, 10
metaphase plates, and at least 10 metaphase spreads were analyzed
under the fluorescence microscope (>30 metaphases).
Mitotic Index. Chromosome slides were stained with Giemsa (15%
in 0.05 M phosphate buffer, pH 6.8, 15 min). The number of mitotic
figures was counted and the mititoc index was calculated as:
Mitotic index
= number of mitosis per total number of cells analyzed x 100)
At least 1000 cells were analyzed for each sample.
Morphologically Transformed
EGV colony
Culture in 6 well
Dish
Culture in 25 cm2
Flask
Culture in 75cm2
Flask
Culture in
2 75 cm2 Flask
(Split Passage!)
Fig. 1. Diagram of culture conditions for generation of immortal EGV cell
lines. Cells from a single isolated EGV colony were progressively expanded after
trypsinization of one-half of the original EGV colony to two 75-cm2 flasks (split
passage 1). Cells were used for cytogenetic analysis from the portion of the
original EGV colony which remained in the dish, and from passage I or 2 EGV
cells.
741
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CHROMOSOME
ALTERATIONS
CORRELATING
IOO-
100
f
1
,9 10-1
Õ
I 011
3
5
7
9
II
13
15
17
19
21
Passoge Number
Fig. 2. Colony-forming efficiency of EGV cultures as a function of passage
number.
Numerical Chromosome Changes in Early Passages of EGV Cell
Cultures That Became Immortal
All EGV cultures surviving to passage 1 were aneuploid
(Table 1). In three of the five EGV cultures (116, 117, and
124), the modal chromosome numbers were between 2n and
3n. Two EGV cultures, 401 and 404, were highly aneuploid
and their modal chromosome numbers were between 4n and
In. Every EGV culture exhibited a numerical increase of chro
mosome 7 as a common abnormality. Numerical increases in
chromosomes 4 and 11 also occurred in all five EGV cultures
as either a common or recurrent abnormality.
Structural Chromosome Changes in Immortal RTE Cell Lines
Nonrandom structural changes involving chromosomes 1, 3,
7, or 15 were observed in all five EGV cultures (Table 1). Four
out of five cultures had structural changes involving the long
arm of chromosome 1 (Iq). EGV 117 had a partial deletion of
Iq (Fig. 3); EGV 124 contained two translocations involving
Iq (Fig. 4); EGV 401 also contained a translocation of Iq (Fig.
5); and EGV 404 had a duplication of Iq (Table 1). The
breakpoints were between q21 and q51 in every alteration.
Three types of structural changes involving the long arm of
chromosome 3 (3q) were observed in four out of five cell lines.
Isochromosome 3 [i(3q)] occurred in EGV 116, 117, and 124
(Figs. 3, 4, 6). Translocations of 3q also occurred in EGV 116,
WITH PROGRESSION
TO IMMORTALITY
124, and 401. In all three EGV cultures with translocations
involving chromosome 3, the chromosome 3 breakpoint was
within ql.
Chromosome 15 contributed to translocations involving 3q
in two of the lines, EGV 116 and EGV 401 (Figs. 5 and 6) and
also formed a translocation with the long arm of chromosome
7, which could be found commonly in three of the EGV cultures
(116, 117, and 124). Another alteration involving chromosome
15 was the isochromosome of the long arm which was observed
in EGV 401 (Fig. 5). The breakpoints of the individual struc
tural changes were unclear.
Three EGV cultures (116, 117, 124) contained specific
marker chromosomes (Figs. 3, 4, 6). The banding pattern of
individual marker chromosomes was similar among all three
cultures, however, it is unclear whether the individual marker
chromosomes had the same origin.
Cytogenetic Findings in Primary MNNG-transformed Colo
nies That Did Not Become Immortal. It was important to analyze
whether or not cells in the EGV cultures which did not become
immortal contained the same chromosome abnormalities which
were found in cells of the immortalized cultures. We analyzed
individual karyotypes of the 29 primary transformed colonies
that did not become immortal. Twenty out of 29 of these
colonies had a normal diploid (42, XY) karyotype (Table 2).
The remaining nine colonies showed karyotypic abnormalities.
Only random gains or losses of chromosomes occurred in cells
from these colonies. None of them showed a gain of chromo
some 7 (which was observed in every established cell line). The
gain of a chromosome 4 or 11 was observed in only a single
colony each, whereas these chromosome abnormalities were
found as common or recurrent abnormalities in every immortal
cell line.
Seven out of the 29 colonies that did not become immortal
showed structural abnormalities involving chromosomes 1, 2,
3, 4, 12, and 14 (Table 2). Only two colonies (EGV 310 and
313), contained a structural change involving Iq. However, the
breakpoints in these aberrations were beyond qSl and different
from the one involved in the structural changes of Iq that were
found in the immortalized cell lines. Isochromosome 3 (i(3q)]
occurred only in EGV 305, and with a low frequency (43%) in
EGV 405.
Cytogenetic Findings in Primary MNNG-induced Transformed
Colonies Which Gave Rise to Immortal Cell Lines
Both numerical and structural abnormalities were found at
early passages of EGV cultures that gave rise to immortal cell
lines (see above), which were not observed in nonimmortalizable
colonies, suggesting that these chromosome changes could be
correlated with the immortalization process. Our next question
Table 1 Cytogenetic findings in MNNG-induced transformed RTE cell lines
lineEGV-116
Cell
chromosome
no.5656
EGV-117
PI
124EGV-40U
EGV-
PIPIPIModal
EGV-404"PassageP2
abnormalities+2,
chromosome
+Y,+t(?;3q),
-3, +4, +7. +13.
+t(15?;7q)
+2, -3, -3, +4, +7, +9, +13,
+i(3q), +t(15?;7q)
5498ND94-135Common-3, -3, +7, +9,+ 11, +Y,
+i(3q), +t(?;3q); +t(4q:lq).
+l(15?;7q)
-1, -3, +4. +4. +6. +7, +8. +9. -10.
+11, +12, +13, +13, -14, -16, -16, +Y,
+t(lq;6q), +t(15?;3q), +i(15q)
+2, +3, +4, +5, +6, +6,
+7.+
11, +11, +11, +12, +17, +18, +19, +20,
+dup(lq), +dup(lq)Recurrent
abnormalities+9,
chromosome
+11, +17, +18,
+i(3q). +t(15?;3q)
+
+Y,+del(lq)
11,
19.
+4, +16,+
* Deviation from tetraploidy.
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CHROMOSOME
ALTERATIONS CORRELATING
I
WITH PROGRESSION
i(3q)
del(lq)
TO IMMORTALITY
3
IM
Fig. 3. Q-Banded karyotype showing a 55,
XYY, +2, -3, +4, +4, +5, -6, +9, +11, -12,
-Hi3, +19, -1-20,+del( 1q), +i(3q), +2t( 15?;7q),
-t-M1 in EGV-117 cell line at passage 1.
7
t(15?;7q)
X
EGV-II7,
Y Y
P.
Fig. 4. Q-Banded karyotype showing a 52,
XY, -1, -3, -3, +4, +7, +9, -12, +13, +16,
+ 17, +18, +19, +t(4q;lq), +t(?;lq), +i(3q),
t(?;3q), +t(15?;7q), +M1 in EGV-124 cell line
at passage 1.
was when during this process did these chromosome changes
occur?
Numerical Chromosome Changes. Every immortalizable pri
mary transformed colony analyzed was aneuploid (Table 3). In
three of the primary colonies (EGV 116, 117, and 124), the
individual modal chromosomes numbers were similar to those
of early passages of the immortalized cultures; between 2n and
3n. EGV colony 401 was heterogeneous with the chromosome
number ranging between 3n and lOn; EGV colony 404 was
near tetraploid in contrast to the heterogeneous ploidy observed
in early passages of the immortalized cultures. The three types
of nonrandom numerical chromosome changes which were
observed among the immortalized cell lines, i.e., the gains of
chromosomes 4, 7, and 11, were not always observed in the
original EGV colonies (Table 3 and 4).
Structural Chromosome Changes. Structural changes involv
ing chromosomes 1, 3, 7, and 15 were common or recurrent
abnormalities in the immortalized cell lines. Most of these
743
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CHROMOSOME ALTERATIONS CORRELATING WITH PROGRESSION TO IMMORTALITY
/t(lq;6q)
3
Fig. 5. Q-Banded karyotype showing a 99,
XXYYYYY, -1, +4. +4, +6, +7, +8, +9, -10,
+ 11. +12, +13, +13, -14, -15, -16, -16,
+ 19, +20, +l(lq;6q),
+t(15?;3q), i(15q),
+M2, +M2 in EGV-401 cell lineal passage 1.
t(15?;3q)
MM*
.
.
•¿
Iti
-; ;
« .
•¿
.
15
i(lSq)
Itti
MM*
XX Y Y Y YY
EGV-401,P.
t(15?;3q)
V' i fi
t(?;3q)
t
Fig. 6. Q-Banded karyotype showing a
57, XYY, +2, -3. -3, +4. +4, +7, +7, +9,
+11, +13, +16, +17, +18, +19, +l(?;3q),
+t(15?;3q), +t(15?;7q). +M1 in EGV-116cell
line at passage 2.
7
t(15?;7q)
II
- *
12
«A
X YY
EGTT-II6,P2
alterations (del(lq), t(?;lq), t(lq;6q), t(4q;lq), i(3q), t(?;3q).
ti 15?;7q)] were also observed in the primary EGV colonies from
which the cell lines arose, with a frequency similar to that
observed in early passages of the immortalized cultures (Tables
1 and 3). The incidence of the other structural changes were
different in the original colonies and early passages of their
respective immortalized cultures.
Correlation of Specific Chromosome Alterations with the Acqui
sition of Indefinite Growth Capacity
Statistical analysis of chromosome alterations which corre
lated with the acquisition of indefinite growth capacity was
performed using the Fisher exact test (see "Materials and
Methods"). Using this statistic, structural alterations in chro
mosomes 1 (P = 0.0035), 3 (P = 0.0015), 7 (P = 0.0017), and
744
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CHROMOSOME
ALTERATIONS
CORRELATING
WITH PROGRESSION
TO IMMORTALITY
15 (P = 0.0001) showed a significant correlation with the
immortal phenotype. Analysis of numerical alterations which
also correlated with the immortal phenotype indicated that +4
(P = 0.00002), + 7 (P = 0.0000004), and +11 (P = 0.00002)
were also associated with the immortal phenotype. In all cases
(except chromosome 3), the numerical alteration was a gain of
the chromosome indicated. Interestingly a numerical loss of
chromosome 3 that occurred in four of the immortal EGVcultures was accompanied by a concomitant structural altera
tion involving 3q resulting in disomy or trisomy for this region
of chromosome 3.
7T7iii
Comparison of M ¡lotieIndex of Immortalizable and Nonimmortalizable Primary EGV Colonies
3i1j18E1f|i11sS;^.CfiILs:••*.5tgZ^l1Ce1nosomes
ivi'o1
The mitotic index of cells from nine primary EGV colonies
that
•¿1o1sunOEI"oC.2did not become immortal and the five primary EGV
colonies from which the five cell lines were derived was deter
mined (Table 5). The average mitotic index of the EGV colonies
that became immortal was 5.36 ±3.01 whereas the mitotic
index of the EGV colonies that senesced was 1.98 ±0.89. Thus,
the mitotic index of the EGV colonies that became immortal
was significantly higher (P < 0.005) than that of those EGV
colonies that senesced.
+
11
CSOo6>•Xoooor--C^V)—fi—-oOS00r-«in^fiIN^1o£>«•o
+'
7i-pÕN
DISCUSSION
Of the 34 transformed colonies isolated, five became immor
tal and the remaining 29 colonies ceased to proliferate prior to
split passage 1. All five colonies that became immortal were
aneuploid. Three types of numerical changes were identified
which were found nonrandomly at early passages of the cultures
that became immortal; gains of chromosomes 4, 7, and 11.
Every culture that gave rise to immortal cell lines had gained a
copy of chromosome 7 as a common abnormality, however,
EBocC5NHiEShromosomeÃ-joj•En•1rio¿
only one primary colony had trisomy 7 as a common abnor
mality (Table 4). This could also be demonstrated for chromo
some 4. Every immortalizable culture had gained a copy of
chromosome 4 as a common abnormality or recurrent abnor
mality. However, only one primary transformed colony showed
+4 as a common abnormality (Table 4). Acquisition of an
additional copy of chromosome 11 was found in all five EGV
cultures that became immortal as a common or recurrent ab
normality, but in the original primary colonies, only two con
tained + 11 (Table 4). These results suggest that numerical
changes in these chromosomes may occur during the immor
talization process.
We found structural alterations involving chromosomes 1, 3,
7, and 15 occurred nonrandomly among the five immortalized
cell cultures. There were five types of structural alterations
involving the long arm of chromosome l(lq); del(lq), dup(lq),
t(?;lq), t(lq;6q), t(4q;lq). These alterations occurred in four
out of five of the cultures which became immortal. Different
EGV cultures had different alterations, but in three out of the
four EGV cell cultures that had alterations in Iq, the alteration
was observed in the original EGV colony (Table 4). The re
olia
maining immortal culture had dup(lq) which was found in 7
b2*5
^**
out of 56 cells from the original colony, suggesting that the
'«¡11§o|•S
o
small subpopulation that had dup(lq) increased during the
immortalization process. In all cases, the alteration in Iq re
sulted in a net gain of Iq, and the breakpoints of the individual
•¿Â£fa
C
alterations were always between q21 and q51 and could be
i0
observed in the primary EGV colony from which the immortal
+ E1
cell line arose.
We found three types of structural alterations involving the
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[i]ÕHCtf1aBi0CMflII
745
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CHROMOSOME
ALTERATIONS
CORRELATING
Table 3 Cytogenetic findings
lineEGV-116
Cell
5554ND62-201
EGV-404"Modal
primary
TO IMMORTALITY
transformed
colonies
abnormalities+2, chromosome
abnormalities+t(15?;7q)
+4, +13, +17, +i(3q), +t(?;3q).
-3, -3, +7, + 11, + 13, +Y, +i(3q): +t(15?;7q)
+2,
+del(lq)+t(lq;6q)
-I, -3, -3. +17. +i(3q), +t(?;3q). +t(4q;lq).+
+t(l-3,
5?;7q),Recurrent
chromosome
no.54
EGV-117
EGV124EGV-401"
WITH PROGRESSION
in MNNG-induced
chromosome
11, +20, +Y
+ 12, +12
88Common
' Deviation from tetraploidy.
Table 4 Chromosomes involved in numerical and/or structural changes in MNNG-induced primary transformed colonies and cell lines
Chromosome number
Passage
EGV-116
Primary
5
123
6
q+
q
q+
(q-)
q+
2-
q+
(q-)
q+
P2
EGV-117
Primary
PI
EGV-124
Primary
PI
7
q+
q+
EGV-401
Primary
2
q+
2q+
9
10
11
12
13
14
q+
q+
17
18
19
20
»q+
*q+
*q+
q+
q+
16
*q+
q+
q+
(+)
q+
15
*q+
q+
q+
PI
8
*q+
2+
-
2*q+
EGV-404
Primary
2+
3+
(C+)
PI
q+
+
" +, gain of a whole chromosome; q+, rearrangement of a long arm of a chromosome; -, loss of a whole chromosome; q-, deletion of a long arm of a chromosome;
*q+, rearrangement of a long arm or centromeric region of a chromosome; C+, rearrangement of the centromeric region of a chromosome; ( ), recurrent abnormalities;
no parenthesis, common abnormalities.
Table 5 Mitotic index (%)
lines is accompanied by a loss of chromosome 3, resulting in a
loss of 3p may be a significant event in the
immortalization process.
Nonrandom structural alterations in chromosomes 7 and 15
also occurred in the cell cultures that became immortal. Three
out of five of the cultures contained t(15?;7q) with a high
frequency (>90%). The original colonies from which they were
derived also had t(15?;7q). Although we could not determine
the exact breakpoint of 7q in t(15?;7q), it was in the proximal
portion of 7q. Another structural alteration involving chromo
some 15 was i(15q), which was found in one immortal cell
long arm of chromosome 3(3q); i(3q), t(?;3q), and t(15?;3q).
culture with a high frequency. In the original colony, i(15q)
Four out of five EGV cultures that became immortal had some could not be found in any of 50 metaphases examined. There
structural changes involving 3q. Sometimes, the same altera
fore, in contrast to t(15?;7q) which occurred in the primary
tions were found in different cultures. The breakpoints of t(?;3q)
EGV colonies, i(15q) and [t(15?;3q)] may have been generated
and t(15?;3q) were within ql. The t(?;3q) was also found in during the immortalization process.
original colonies of the immortalizable cultures that contained
In general, the structural alterations that correlated with the
this translocation. However, t(15?;3q) was not found or found
immortal phenotype occurred very early during transformation
only in a small population of the original colonies, suggesting
and were detectable in the primary transformed EGV colonies.
the t(15?;3q) occurred in a minor subpopulation, which in
Detection of structural alterations at very early stages is con
creased during the immortalization process. The i(3q) aberra
sistent with the hypothesis that these changes were induced by
tion observed may have occurred by centric fusion, and if so, MNNG, which is clastogenic and induces chromosome aber
i(3q) is genetically equivalent to disomy 3. It is also interesting
rations in RTE cells (5). In most instances, numerical altera
tions which correlated with immortalization appeared to occur
to note that the increase in 3q in four of the five immortalizable
746
in
mitosis(%)1.41.03.21.41.30.92.83.12.7
in
mitosis(%)4.72.33.46.410.0Nonimmortal
Immortal
net loss of 3p. This
EGV colonies105107108111122214305313405Cells
EGV colonies116117124401404Cells
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CHROMOSOME
ALTERATIONS
CORRELATING
later during the immortalization process during successive pas
sages in vitro.
Several cellular oncogenes have been localized to rat chro
mosomes 1, 3, 4, 7, and 15. H-ras has been mapped to chro
mosome 1 (11), the long arm of chromosome 3 (3q) contains
c-abl (11), K-ras and c-ra/are located on chromosome 4 (12),
c-myc and c-sis are located on chromosome 7 (11, 12), and
erbA.-l is on chromosome 15 (11). Of these, the expression of
H-ras, K-ras, c-raf, and c-abl has been quantitated in trans
formed RTE cells. Only modest increases (3-5-fold) in the
expression of these genes was observed (13).
Numerical changes in chromosomes 4 and 7 have been ob
served during transformation of rat cells in other systems.
Additional copies of chromosome 4 was a consistent abnor
mality observed in ethylnitrosourea-induced
neurogenic tumor
lines of the rat (14-16), and trisomy of chromosome 7 has been
observed in RSV-induced rat sarcomas (17). Structural altera
tions of chromosomes 1, 3, and 7 have also occurred with a
high frequency in other rat transformation models. In tumors
induced in rats with 3-methylcholanthrene or 3,4-benzpyrene,
chromosomes 3, 4, 7, and 11 were most frequently engaged in
marker formation, and the long arm of chromosome 1 and 2
existed as trisomies (18, 19). In rat cell lines transformed in
vitro by 7,12-dimethylbenz(a)anthracene
and in tumors induced
with this carcinogen, chromosomes 1 and 3 were trisomie and
frequently involved in marker formation along with chromo
some 7 (20, 21). Hypersomy of chromosomes 1 and 3 has been
described in rat cells transformed by adenovirus (human) type
12 (22), and rat embryo cell lines transformed by herpes simplex
viral DNA, chromosomes 3 and 7 were frequently involved in
the formation of marker chromosomes (23). In some cell lines
derived from tumors produced by these cells, chromosome 15
was involved in the formation of marker chromosomes. Struc
tural alteration of chromosome 7 has also been noted in spon
taneous rat immunocytomas (24). In addition, earlier studies in
our laboratory indicated that aberrations of chromosomes 3
and 4 were recurrently observed in primary EGV colonies (5).
Thus the alterations of certain chromosomes, especially chro
mosomes 1, 3, and 7, and their involvement in marker forma
tion in rat cells transformed by chemical carcinogens or viruses
may be significant in the selective survival and proliferation of
transformed cells and tumor progression regardless of tissue
origin and etiological agents.
ACKNOWLEDGMENTS
We thank Drs. F. Miller and O. Moss for their help in the statistical
analysis of these data, D. Rusnak for excellent technical support, and
S. Leak and L. Smith for preparation of this manuscript.
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747
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Nonrandom Chromosome Alterations That Correlate with
Progression to Immortality in Rat Tracheal Epithelial Cells
Transformed with N-Methyl-N′-nitro-N-nitrosoguanidine
Sumiyo Endo, Paul Nettesheim, Mitsuo Oshimura, et al.
Cancer Res 1990;50:740-747.
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