[CANCER RESEARCH 33, 3250-3258, December 1973]
Chromosomal Banding Patterns and in Vitro Transformation
Syrian Hamster Cells
of
J. A. DiPaolo, ' N. C. Popescu,2 and R. L Nelson
Cyiogenetics and Cytology Section, Biology Branch, Division of Cancer Cause and Prevention, National Cancer institute, Bethesda, Maryland 20014
SUMMARY
Chromosome banding techniques were used to analyze
the chromosomal constitution of hamster cells transformed
by chemical carcinogens and fibrosarcomas obtained after
injection of the transformed cell lines. Each transformed
line is considered unique because each was derived from
fetal material from a different pregnant animal. The chro
mosome modes of the transformed lines and tumors were
generally near-diploid. Analysis of bands verified the oc
currence of identical banding patterns in the marker chro
mosomes of transformed lines and tumor-derived cultures,
thus providing unequivocal evidence that the fibrosarcomas
were produced by the cells that were transformed in vitro
and making it possible to recognize 10 marker chromo
somes and their origin. Different carcinogens may produce
transformations associated with the same specific marker,
but not all transformed lines have the same markers even
with the same chemical carcinogen. Some markers were
found in the tumor-derived culture and not the transformed
line or occurred in later passages of both the transformed
line and tumor cultures but not in the early passage of
these lines. In some transformed cell lines the chromosome
number increased but did not involve a specific chromo
some group or pair. The incidence of chromosomal devia
tion in cancer is species dependent and contributes to the
characteristics of cancer. Chromosome alterations are re
sponsible for the genotypic changes capable of autonomous
growth and thus are considered reflections of secondary
alterations. The question whether chromosome changes are
the causal factors in carcinogenesis remains unanswered.
INTRODUCTION
The role of chromosomal changes in carcinogenesis has
been debated since the time of Boveri (4) who suggested
that a specific type or types of alterations in chromosome
constitution were responsible for the new morphology (neoplastic) of the cell and the concomitant changes in biochem
ical behavior that lead to apparently unregulated growth
'To whom reprint requests should be addressed, at National Cancer
Institute, Building 37, Room 2A13, Bethesda, Md. 20014.
1Visiting Associate, National Cancer Institute, NIH. Permanent
address: Oncological Institute, Bucharest, Romania.
Received June 27, 1973; accepted September 18, 1973.
3250
and multiplication. Conflicting interpretations of chromo
somal changes seen during in vitro transformation of cells
induced by chemicals and in vivo experiments, as well as
in studies on human cells, indicate the complexity of the
problem and the frustration in attempting to develop in
clusive theories.
Chromosome analysis by a conventional Colcemid tech
nique of chemically or virally induced tumors or trans
formed lines of the Syrian hamster has resulted in the con
clusion that no detectable universal changes exist and that
the chromosome modes may range from hypodiploid
through hypertetraploid (7, 10, 13, 31, 33). The chromo
some alterations were considered minimal and of a random
occurrence that was independent of the transformation (13).
The use of banding techniques makes possible the identi
fication of each pair of chromosomes of the Syrian ham
ster karyotype so that numerical changes in chromosome
pairs and any uncommon banding patterns that occur in
chromosomes in association with transformation may be
accurately analyzed (34). In this study chromosomes of 9
transformed cell lines, each originally derived from a dif
ferent animal, as well as a number of tumors obtained by
injecting the transformed cells, are analyzed for numerical
and structural changes in terms of their bands.
MATERIALS
AND METHODS
Cell culture procedures and the transformation assays
have been described (11, 13). Transformed cell lines were
developed from isolated, carcinogen-treated, transformed
colonies. Each cell line originated from embryos of different
hamsters. When cells from the transformed lines were in
jected into hamsters, fibrosarcomas were produced at the
site of injection (12, 13). The tumors were returned to cul
ture and are referred to as tumor-derived cultures (t).
Cultures for chromosome study were grown in Falcon
Petri dishes in Dulbecco's modification of Eagle's medium
supplemented with 10% fetal bovine serum, in a humidified
10% CO2 incubator. Chromosomes of tumor cells were ex
amined from a 48-hr-old primary or 24-hr-old secondary
culture. Cells were harvested after 4 hr of Colcemid incuba
tion (0.08 ¿ig/ml),treated with a 1:2 solution of medium
and distilled water for 14 min, and fixed in methanol: glacial
acetic acid (3:1). Slides were prepared by the air dry chro
mosome technique. The banding pattern of the hamster
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Chromosome Band Patterns
chromosomes was established by 3 different techniques:
modification of the acetic saline Giemsa technique
(34) of Sumner et al. (43), the trypsin technique of Seabright (39), and the fluorescent technique of Caspersson
et al. (5) as modified by Francke and Nesbitt (15). Wellspread metaphases with banding patterns were photo
graphed. Karyotypes were prepared in accordance with
the banding schematic idiogram for Syrian hamster cells
(34).
RESULTS
Nine different transformed cell lines obtained after treat
ment with BP,3 AFB,, PS, or 4NQO and 6 tumor-derived
cultures were examined. One cell line, AFB,39, and the
corresponding tumor culture were examined at 2 different
passage levels. The majority of the transformed cell lines
(8 of 9) and the tumors (4 of 6) have near-diploid chromo
some modes (Table 1). Where both the transformed line
and corresponding tumor were examined, the stem-line was
similar. The distribution of the markers in cell lines range
from no marker, regardless of the banding technique used,
to as many as 4 markers (Table 1). The 21 pairs of autosomes and 2 sex chromosomes that constitute the normal
hamster complement are clearly identifiable. In some
transformed lines, no abnormal chromosomes were found
and the modal chromosome number was 44. In these cases,
the stem-line was near diploid and, although trisomy ex
isted, the trisomy did not always involve a specific chromo
some. For example, 4NQO17 has a major near-diploid
mode and no chromosome marker but may have extra
chromosomes belonging to B5 and C15 (Fig. 1); 4NQO17
also has a minor hypertetraploid mode, and in the cell
shown (Fig. 2) the extra chromosomes belonged to Groups
B5 and BIO. Thus all chromosomes are identifiable as to
specific pair.
On the basis of band analysis, the derivation of 10 marker
chromosomes (p and q signify short and long arms, respec
tively) found thus far, is: M,, t(A2p; ?); M2, t(D16q;
D17q); M3,t(C15p; F21); M<, del(Alq); M5, t(Aq;?); M.,
t(Xq; ?); M7, t(Alq; A2q); Ma, t(A4p; ?); Mb, t(C15q; ?)
(Figs. 3 and 4). In time, it will be possible completely to
substantiate the derivation of the markers.
MI, the most common marker in this series, is shown
next to a pair of normal A2 chromosomes followed by the
schematic representation of the M t and A2 (Fig. 3). The
additional chromosomal material in the A2q arm required
either an unequal translocation to produce a longer q arm
or an insertion of a new piece of chromosome in the lower
end of the q arm. Areas similar to the new area are found in
Chromosomes Al, C15, and D17. In some cells, the pres
ence of M i is accompanied by a loss of 1 Al, but in others
such as in Fig. 5 both Al chromosomes are present. The
marker is never associated with a loss of D17 and, in fact,
D17 often occurs as a trisomy. The most likely origin of the
translocation in M t is the distal portion of the long arm of
C15, which is always monosomic when M , or M 3are pres
ent. Conversely, when a pair of CIS's are present neither
marker is found. M ! was found in 4 transformed cell lines
and in 4 of the 6 tumors. This marker as well as the others
occurred independently since each transformed cell line
originated from a different animal. The incidence of M,
Table 1
was high; it was 80% in 1cell line. Interestingly, M , appears
Chromosome characteristics of transformed cell lines and tumorin 4NQO16t, whereas 4NQO16 has no markers; BP29 had
derived cultures
no markers, but 1 cell of BP29t had 1 M,. AFB,39, when
examined at the 6th passage, and its corresponding tumor
lineBP45BP45"BP28BP29BP29tAFB,
Cell
no.42424344444445444443448888444445Markers"1,
had only Marker M3 at Passage 13; the transformed line
and the corresponding tumor had M3 and M, as well as
4641-4842-4641
31,2,
other markers. M3 also occurs in a nonrandom fashion,
31,2NoneIa331,3,4,1,4,
2,
since it appears in 3 different transformed cell lines and in 2
4541
different tumors. Two markers identified only in tumor
4542-4542-4643-4542-4642
4NQO16t and in no transformed line are indicated by the
39AFB,39tAFB.39AFB,39tAFB
letters a and b.
An example of a quinacrine mustard preparation from
BP45 (Fig. 5) shows that, in addition to multiple markers,
51,3None6,
,42PS14PS66PS66t4NQOI64NQ01614NQO17Passage1313g271132168122726Stem-line43
4643-4680
new chromosomes may be present such as the Di, a dicentric that is in part made up of a C12 and C13 as well as an
8879
77None1,
unidentified chromosome piece. One chromosome of Pair
8843
B7, C12, C15, F21, and Pair C13 were missing. Some of
4643
4642
a,None5b
3,
these occurred as portions of new marker chromosomes.
46Modal
The total number of chromosomes in the D group has in
" Markers verified by the study of banding patterns in 20 karyotypes
creased consistent with previous observations.
A karyotype (Fig. 6) with 44 chromosomes from
as well as in the metaphases.
"Tumor culture derived from corresponding transformed line; mini
4NQO16t tumor exhibits trisomy for B7 and D17 and
mum of 50 metaphases used to obtain stem-line and modal number.
monosomy for Al, C15, and F21. Furthermore, in addition
c Marker found in only 1 cell.
to the M, marker, 2 markers, Ma and Mb, occurred only
in Tumor 4NQO16t. The Ma and Mb markers result from
'The abbreviations used are: BP, benzo(a)pyrene; AFB,, aflatoxin B,;
translocations that have been added to the terminal por
PS, propanesultone; 4NQO, 4-nitroquinolone /V-oxide.
tions of the A4p and C14q arms, respectively. The trans-
DECEMBER
1973
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3251
J. A. DiPaolo, N. C. Popescu, and R. L. Nelson
location to the short arm of the A4 chromosome is stained
uniformly dark.
The ability to analyze all the chromosomes on the basis
of their banding patterns made possible the examination of
a number of lines for changes in number of chromosomes
constituting each identifiable pair (Table 2). The increase
or a decrease in certain chromosomes is random. In fact, in
PS66 and PS66t, which have tetraploid modes, occasionally
one of the X chromosomes was missing. No significant in
crease, as indicated by the blocked number, was found
among the transformed lines or tumor studied; however, in
Tumors BP45t and 4NQO161, trisomy for D17 was present
in at least 16 of 20 karyotypes. Both tumors as well as the
transformed line BP45 were monosomic for CIS; the other
C15 had been utilized in the formation of M ¡and M3. It
can be generalized that no increase or decrease in a specific
chromosome number was associated with any of the trans
formations.
DISCUSSION
Chromosome banding techniques have permitted addi
tional description of the role of chromosomes in carcinogenesis (26, 34). These newer techniques represent a power
ful tool since, as demonstrated in this study, numerical
changes in chromosome pairs and all chromosome markers
become identifiable. Syrian hamster cells are mainly diploid
in constitution with 1 to 2% in the tetraploid range (13).
Carcinogen treatment results in transformed lines usually
near-diploid but occasionally with tetraploid modes; injec
tion of these cells into animals results in corresponding tu
mors with similar chromosome modes, karyotypes, and
markers with identical banding patterns.
Some transformed lines have specific markers that can
be cataloged according to the uncommon banding pattern
of the chromosomes. The occurrence of the identical marker
in the transformed cell line and the resulting fibrosarcoma
are definite indications that the cells that underwent trans
formation were responsible for the tumor formation. Some
markers, particularly MI and M3, occurred as a result of
treatment with different carcinogens; however, not all the
transformed lines had the same markers even when trans
formed with the same chemical. Marker M,, which was
observed most frequently in the transformed lines and tu
mor cell cultures, was also found in 1 case in a tumor but
not in the parental transformed line. The presence of
Marker M , in a later passage of a transformed cell line and
tumor as opposed to its absence at the 1st examination is an
indication that changes at the chromosomal level are sec
ondary. Therefore, although the specific chromosome
change is considered to be nonrandom, it cannot be consid
ered universal. The possibility that M, is polymorphic for
A2 was considered because the mechanism for the forma
tion of this specific chromosomal translocation is not clear
and because the arm ratio of A2 (2.5 to 2.8) may vary (24).
Reexamination of chromosomes of control cells prepared
for bands has failed to show any deviation from the band
ing pattern previously reported (34). Marker 3, although
not as common as M ,, occurred repeatedly and thus can
be considered nonrandom.
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CANCER RESEARCH
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VOL. 33
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Chromosome
Marker M ¡found in the current study probably also oc
curred in other carcinogenesis studies prior to the advent of
banding techniques (10, 13, 31, 33). Reports stating that
E20 occurred as a trisomy in tumor cells (6, 8, 31, 33) may
be in error since only by banding procedures can M3 be
shown to originate from CIS and F21. In studies on tumors
from rats (23, 28, 36, 42) and Chinese hamsters (29), identi
cal marker chromosomes were found by means of old tech
niques. However, reconciliation of older with current
chromosome data is difficult. For example, a number of
apparently similar chromosome aberrations found in hu
man lymphoblastoid cell lines have been shown with the
quinacrine fluorescent technique to be not identical (40).
Human primary tumors do not show a consistent chromo
some pattern (1, 21, 25, 38) except for chronic granulocytic
leukemia which possesses the unique Ph1 chromosome in
nearly every case (32). Recently, the quinacrine banding
technique has been used to identify an addition of dully
fluorescing material at the end of the long arm of 1 Chro
mosome 9 (37); the amount of added material is approxi
mately equal to the amount missing from the Ph1 chromo
some. Thus the possible existence of a translocation would
suggest that at the chromosomal level cancer may occur
without any net change of genetic material.
Boveri's concepts of chromosomal imbalance, including
Band Patterns
in check" (18). Thus, both groups of investigators suggest
that new chromosome combinations may lead to the sup
pression of cancer.
When 2 cells are fused, a new cell has been formed that
has, in addition to the 2 sets of chromosomes, other charac
teristics of the nuclei and cytoplasm. For example, when
human mouse hybrid cells were analyzed for rRNA synthe
sis, only the mouse type 28 S could be detected even in hy
brids with up to 35 human chromosomes per cell; a plau
sible explanation is that the transcription of human ribosomal genes is repressed in hybrids (14). The changes in the
degree of malignancy will have to be correlated with
changes in properties of cell hybrids.
The chromosome evolution of tumors (22) and the finding
that many primary cancers may consist mainly of cells with
a diploid number of chromosomes (2, 9, 19) suggest that
the observed chromosome changes are not necessarily asso
ciated with cancer. For example, in the case of chemical
carcinogenesis utilizing in vitro systems, gene regulation
may play an important role. Variants represent responses
of cells to different environments, and the variability in the
deviation from the diploid status indicates that a number of
stem-lines are possible. Cells subjected to a new environ
ment are perpetuated by genetic readjustment. Environ
mental changes will require different numbers of cell in
the abnormal chromosome content of the cell as the under oculum for tumor formation in adult animals but will not
lying cause of neoplasia, continue to challenge investigators effect absolute reversal of the tumorigenic properties of
of cancer. In some hamster transformed cell lines, the the cells.
chromosome number increase did not involve a specific
Normal development from a fertilized egg results in
chromosome group or pair. Gofman et al. (16, 27) using a diverse cell types that constitute a variety of different or
semiautomatic chromosome analysis system expressed the gans in which no change in chromosomes is required. The
imbalance as "abnormal ratio of chromosomes of particular growth of an entire carrot from 1 parenchymal cell (41) or
type to chromosomes of another type, in contrast to the a fully developed normal adult frog (17) from the nuclear
analogous ratio in the normal human diploid cell." Rééval
transplantation from an adult intestinal cell to an enucleated
uation of these results with the same approach failed to frog ovum are evidence that changes in states of differenti
substantiate these findings because the average number of ation can occur without any chromosomal changes. The
chromosomes of each type in a population of cells had been structural and numerical changes that occur after trans
counted (3) rather than the absolute number of chromo
formation depend upon the species and are part of the
somes in each individual cell. A similar computer method problem involving cell differentiation and the evolution of
was used by Muldal et al. (30), who found in humans a sig cell populations to autonomy.
nificant gain in Group C and losses in Groups D and G.
The finding of DG translocations has not been verified.
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Chromosome Band Patterns
Fig. 1. Karyotype of a cell from a 4NQOI7-transformed cell line. This line has no detectable structural modifications and all chromosomes have a
normal banding pattern (trypsin preparations). The karyotype consists of 46 chromosomes; trisomy or B5 and CIS is present.
Fig. 2. A hypertetraploid cell from the same 4NQO17-transformed cell line. According to banding patterns, all chromosomes are identifiable as
normal. There are 90 chromosomes, with the 2 extras belonging to Groups B5 and BIO.
Fig. 3. Enlargement of M , and a pair of normal A2 chromosomes (acetic saline Giemsa preparation) followed by schematic representation of Marker
1 and the A2 for comparison. This chromosome marker was obtained from a 4NQO16t cell, and it can be seen that, following the largest negative zone
of the long arm, an additional 2 positive bands have been inserted.
Fig. 4. Representatives of all markers found subsequent to M ,; M2through M7 occurred in cell lines and/or tumor cultures, whereas Ma and M„¿were
found in only 1tumor. The new chromosomes are either between their normal components orto the left of their normal analog.
Fig. 5. A karyotype of a subdiploid cell from BP45-transformed cell line (quinacrine mustard preparations). The new chromosomes are indicated
by ML M 2, M 3, and Di, as well as trisomy for D17. Missing are l B7, C12, CIS, and a pair of C13 chromosomes.
Fig. 6. Karyotype of a cell from 4NQO16t culture with several markers as well as numerical changes in several different pairs of chromosomes
(acetic saline Giemsa preparation). Groups B7 and DI7 are trisomie and Al, CIS and F2I monosomic; M, and M3 are present as well as M. and Bb,
which were found only in this specific tumor line.
DECEMBER
1973
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3255
J. A. DiPaolo, N. C. Papesca, and R. L. Nelson
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VOL. 33
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Chromosome Band Patterns
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DECEMBER 1973
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3257
J. A. DiPaolo, N. C. Popescu, and R. L. Nelson
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CANCER
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VOL. 33
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Chromosomal Banding Patterns and in Vitro Transformation of
Syrian Hamster Cells
J. A. DiPaolo, N. C. Popescu and R. L. Nelson
Cancer Res 1973;33:3250-3258.
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