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[CANCER RESEARCH 33, 3259-3265, December 1973J
Distribution of Chromosome Constitutive Heterochromatin of
Syrian Hamster Cells Transformed by Chemical Carcinogens
J. A. DiPaolo1 and N. C. Popescu2
Cytogenetics and Cytology Section, Biology Branch, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland 20014
SUMMARY
The utilization of the C band technique for constitutive
heterochromatin has permitted further analysis of both nor
mal and marker chromosomes that were found following
neoplastic transformation and in tumors derived from the
transformed cells. Syrian hamster cells that have been
transformed by chemical carcinogens have chromosomes
with constitutive heterochromatin characteristic of the
species. In addition to the completely heterochromatic
short arms of the submetacentric autosomes, the long arms
of both X chromosomes and E20 are entirely heterochro
matic. Heterochromatin alterations occurred in only 2 dif
ferent tumor cultures and in no transformed lines and thus
are not a causative factor in transformation. The C band
technique facilitated the analysis of marker chromosomes.
INTRODUCTION
When a number of neoplastic transformed lines, each of
which had been obtained in vitro, and several of the corre
sponding tumors obtained from injecting the transformed
cells into animals were analyzed by reproducible Giemsa
(G) or quinacrine (Q) chromosomal banding techniques,
10 markers as well as the individual chromosomes belong
ing to the different chromosome pairs of the Syrian ham
ster karyotype were identifiable (8). It was possible to
determine the origin of the marker chromosomes and to
conclude that in malignant transformation of the chromo
somal changes, including changes in number of chromo
somes as verified by the banding patterns, are reflections of
secondary alterations rather than being causally related
to the cancer.
Procedures now exist for differentiating constitutive
heterochromatin from euchromatin areas (2). Chromo
somes with similar horizontal banding patterns may differ
in their heterochromatin distribution (C pattern). Hetero
chromatin is a complex subject and the concepts concern
ing it are filled with contradictions (5, 19, 21). However, it
is generally accepted that heterochromatin represents
blocks or regions on chromosomes that are tightly con'To whom reprint requests should be addressed, at National Cancer
Institute, Building 37, Room 2A13, Bethesda, Md. 200I4.
2Visiting Associate. National Cancer Institute. NIH. Permanent ad
dress: Oncological Institute, Bucharest, Romania.
Received August 23, 1973; accepted September 18, 1973.
densed, genetically inactive, and in terms of DNA syn
thesis late replicating.
Variations in the amount of constitutive heterochroma
tin per cell have been considered responsible for changes in
phenotypic effects referred to as position effects (20). Chro
mosomal aberrations commonly observed in neoplasia
could involve heterochromatin because of its known sus
ceptibility to breakage by mutagens (14). The direct expo
sure of Syrian or Chinese hamster cells to polycyclic
hydrocarbons has resulted in wide distribution of carcino
gen-associated breaks in different chromosome regions
(3, 13). Although the response to carcinogen may be coin
cidental with areas known to be rich in heterochromatin,
one result accompanying chromosome breakage is lethality
related to concentration of carcinogen (J. A. DiPaolo and
N. C. Popescu, unpublished data). This study presents
observations of heterochromatin distribution of chromo
somes of 9 transformed cell lines and 4 primary tumors
(obtained by injecting the transformed cells) all of which
have been studied in terms of horizontal bands.
MATERIALS
AND METHODS
Heterochromatin of metaphase chromosomes was local
ized by a modification of the technique of Arrighi and Hsu
(2). Chromosomes of 9 in vitro transformed cell lines were
examined at the 7 13 passage whereas the 4 tumor cul
tures were examined at the 1st or 2nd passage subsequent
to removal of the tumor from the animal.
Chromosome DNA replication pattern was studied by
using thymidine-3H incorporation and autoradiography.
Monolayer cultures in log phase were incubated with thymidine-3H (6.7 ci/mM, 1.0 juCi/ml) for 1 hr. The medium
containing thymidine-3H was replaced with fresh medium
containing 0.04 ng colcemid per ml of medium 2.5 hr before
the cultures were harvested and chromosomes were pre
pared. For studies of early replication patterns, the cul
tures were incubated with thymidine-3H for 30 min followed
by treatment with cold thymidine (100-fold concentration)
9 hr before fixation of the cells. Metaphase spreads were
subjected to G chromosome banding technique. After the
metaphases were stained and photographed, the stain was
removed with absolute methanol and cells rinsed with deionized water. Autoradiography was performed with
NTB2 Kodak emulsion for 6 days at 4°.After being de
veloped and fixed, the slides were conventionally stained
with 5% Giemsa in distilled water. The metaphases were
DECEMBER 1973
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3259
J. A. DiPaolo and N. C. Popescu
relocated and the adequately labeled ones were photo
graphed.
regions of the tumor cells. The study of early and late DNA
replication (Fig. 2, a and c) shows that the heterochromatic
portion of the marker has a sequence of replication similar
to that of the sex chromosomes. In addition, the short arm
RESULTS
of Chromosome A4 which is a normally late labeling is free
of label at the end of the S phase (Fig. 2c). A similar situa
Of 9 different transformed cell lines obtained from treat
tion
was associated with the new heterochromatic translo
ment with BP3, aflatoxin B,, PS, or 4NQO and 4 tumors
cation to the B9 chromosome. The origin of marker
examined, abnormal heterochromatin segments were found
chromosomes by ordinary staining procedures is difficult,
in only 2 tumors derived from cell lines transformed by
if not impossible (Fig. 2, b and d).
BP and 4NQO. Karyotypes of BP45t that had been stained
Because of the finding after viral (7) or chemical (16)
with quinacrine mustard showed, in addition to the ex
transformation of extra chromosomes that appeared to
pected banding pattern, a diffuse, fluorescent, translocated
mimic E20, additional documentation concerning M3 is
segment to the short arm of 1 of the B9 chromosomes (Fig.
presented. In Fig. 3, Pair E20 and the marker chromo
la). The occurrence of this characteristic is approximately
some M 3 are compared by conventional staining, DNA
30% and consequently is not considered a marker in the
replication, and C band procedure. The confusion of M3
same sense as are M, and M 3 which were found in more
with E20, which occurs in conventional staining technique,
than 50% of the metaphases examined. In 4NQO16t (Fig.
is resolved by studying the DNA replication pattern or C
16), a marker chromosome, Ma, consisted of a nonbanded,
bands. During late replication the long arms of both E20
darkly stained translocation to the short arm of the A4
chromosomes are heavily labeled but M3 is not labeled. In
chromosome. This marker can be contrasted to Marker
terms of C bands it has been shown previously (Fig. lc) that
Mb which has a banded translocation to the long arm of
in tumor cells the long arms of E20 are heterochromatic but
C14. With the C band technique, normal heterochromatin
that M 3 has heterochromatic regions associated with the
distribution was found in all 4NQO16t chromosomes iden
dual centromeres.
tified as normal by G band technique.
Markers M6 and M7 (Fig. 3) were associated with the
The Syrian hamster heterochromatin chromosome pat
PS-transformed cell line and tumors; both have subtetratern differs from most species in that all the submetacentric
autosomes have short arms that are totally heterochro- ploid modes (8). The C banding procedures provide addi
tional information about markers M6and M,. M 6 involves
matic. In addition, the long arm of both X chromosomes
a translocation to the heterochromatic arm of the X chro
as well as the long arm of the smallest metacentric are en
mosome. After the addition of the translocation to the X
tirely heterochromatic (Fig. \c). Both female X chromo
to form M6, the basic X continues to exhibit the banding
somes exhibit identical G (18) and C band patterns. Further
pattern determined with the G technique and is late in repli
analysis of chromosomes of this metaphase (Fig. \c) yields
cating for its entire length, consistent with the concept that
additional information concerning chromosome markers
the inert X chromosome is involved. Although cells of the
that have been previously identified by the techniques for PS-transformed line and tumor possess 4 X chromosomes,
horizontal bands (G, Q, or trypsin-Giemsa). Because of the
2 of which are late replicating along the entire length, only
uncommon C pattern, it is possible again to detect M,,
1 chromosome possesses a translocation. The translocation
M 2, and M3. The heterochromatic pattern of M i is identi
does not alter the C band distribution of the X chromo
cal to that of an A2, thus confirming that the translocation
some. This is in contrast to the addition of heterochromatic
was euchromatic and autosomal in origin. It was concluded
regions reported above for BP45t and 4NQO161. M7 is a
that the origin of M2 was a result of a centric fusion of a new metacentric marker chromosome with the character
D16 and D17 (8). The C band technique proves that only 1
istic banding pattern of the long arms of Chromosomes A1
centromere is present. By ordinary staining techniques, M3 and A2, neither of which are late replicating. In contrast to
could be confused with Pair E20. The utilization of the C
the metacentric M3, which possessed a double centromere,
band technique shows that, whereas Pair E20 (arrows) has
M 7has retained only 1centromere.
darkly stained long arms, the M3 has only centrally located
heterochromatin. In a metaphase spread from a cell of the
4NQO tumor (Fig. \d), C band preparation shows again DISCUSSION
that one-half of each X consists of darkly stained hetero
chromatin. The only deviation in heterochromatic pattern
Examination of cell lines for heterochromatin has been
occurs in the chromosome marker, Ma. This marker oc limited to chromosomes of a kidney line from an adult
curred as a result of a translocation to the short arm of A4. African monkey (4), BALB/3T3 cells (17), and an aneuA similar type of translocation was found in Tumor BP45t; ploid line derived from Chinese hamster ovary (6). In
however, in that case the translocation occurred to the short BALB/3T3 cells transformed by direct application of
7,12-dimethylbenz(a)anthracene
or /V-methyl-/V'-nitro-./Varm of a B9 chromosome (Fig. \a).
Chromosome replication in the presence of thymidine-3H nitrosoguanidine, the heterochromatin pattern was similar
provides additional information of these heterochromatic
to the control BALB/3T3 line (17). The formation of tu
mors in BALB/c mice provided evidence that the cells
3The abbreviations used are: BP, benzo(a)pyrene; PS, propanesultone; were neoplastic (9). In the current study, the chromosomes
4NQO, 4-nitroquinoline /V-oxide.
of Syrian hamster transformed cell lines and tumors
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Chromosome Constitutive Heterochromatin
that have been previously examined for horizontal bands
were examined for constitutive heterochromatin (8). All
transformed lines had normal constitutive heterochromatin
distribution similar to that described previously by Hsu and
Arrighi (12) and late DNA replication pattern as reported
by Gallon and Holt (10). Also both sex chromosomes of the
female Syrian hamster had identical G band patterns (18);
the current study shows that one-half of each X chromo
some is totally heterochromatic. With thymidine-3H and
autoradiographic technique, only one of the X chromo
somes is late labeling and becomes completely inactive in a
random fashion as suggested by Lyon (15).
In 2 of the tumors studied, BP45t and 4NQO161, hetero
chromatic segments were translocated to the short arm of
Chromosomes B9 and A4, respectively. Such rearrange
ments can be considered of secondary significance repre
senting another type of chromosomal alteration responsible
for in vivo tumor progression. In both cases the translocated
segments have a sequence of late replication similar to the
sex chromosomes. One effect of the heterochromatic translocation to the short arm of B9 or A4 was an alteration of
the replication time of the original short arms which were
no longer late replicating. The alteration in the total amount
of heterochromatin in the genome may be responsible for a
type of position effect resulting in activation of normally
repressed chromosome segments. The origin of this new
heterochromatic region is unknown since the source of such
large segments would normally be the sex chromosomes;
however, in all metaphases and karyotypes, both X's are
present and normal. The most likely possibility is that the
translocated segments were autosomal and euchromatic
and underwent a process of inactivation and condensation.
In another case, there was a translocation to only 1 X
chromosome of the tetraploid PS66-transformed cell line
and tumor. This translocation was not heterochromatic
material but was attached to the totally inert X chromo
some. No change occurred in either the replication time or
the heterochromatic pattern of the involved X chromo
some. In a recent study of chromatin from minimal-devia
tion rat hepatomas, it was concluded that on the basis of
template activity and electrophoretic profiles the major
nonhistone proteins compared with normal liver chromatin
(1). This suggests that some of the difference between the
chromatin of hepatomas and liver may be responsible for
malignant progression rather than reflective of the process
of neoplastic transformation.
Transformation by a variety of chemical carcinogens ap
pears to occur without any changes in the heterochromatin
distribution of Syrian hamster cells. Although chromosome
breaks have been observed in cells following exposure to a
variety of chemical carcinogens with toxic properties
(Refs. 3 and 13; J. A. DiPaolo and N. C. Popescu, unpub
lished data), the constitutive heterochromatin has not been
studied. Changes in constitutive heterochromatin of human
cells produced by toxic concentrations of mitomycin have
been reported to result in lesions in heterochromatin areas
in 1 of 10 cultures (11). Although alkylating carcinogens
may produce the same type of alteration, the likelihood of
their relevance to neoplastic transformation is probably
DECEMBER
minimal since no alteration in heterochromatin was ob
served with such chemicals.
C band analysis provided information concerning the centromeric areas of the chromosomes as well as confirmatory
evidence of some markers that otherwise may be confused
since they mimic other chromosomes. For example, on the
basis of C band and late DNA replication, the origin of M3
was definitely established. M3 has a distinct replication
sequence pattern, C band stain, and 2 centromeres all
consistent with the conclusion that M3 is a result of the fu
sion of the short arms from C15 and F21. Without this
analysis, the origin of M3 might be confused because it has
certain limited similarities to a B9 that has been deleted
below the centromere on the long arm. In M2 and M7,
which consist of the long arms of different chromosomes,
the markers retained only 1 centromere. Thus, C band
analysis furnished additional information concerning 3 of
10 markers first recognized by horizontal band analysis.
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of Some Properties of Chromatin from Two "Minimal Deviation"
Hepatomas. Cancer Res., 33: 1169-1176, 1973.
2. Arrighi, F. E., and Hsu, T. C. Localization of Heterochromatin in Hu
man Chromosomes. Cytogenetics, 10: 81-86, 1971.
3. Benedict, W. F. Early Changes in Chromosomal Number and Struc
ture after Treatment of Fetal Hamster Cultures with Transforming
Doses of Polycyclic Hydrocarbons. J. Nati. Cancer Inst., 49: 585 590.
1972.
4. Bianchi, N. O., and Ayres, J. Heterochromatin Location on Chro
mosomes of Normal and Transformed Cells from African Green
Monkey (Cercopithecus aethiops). Exptl. Cell Res., 68: 253-258,
1971.
5. Comings, D. E. The Structure and Function of Chromatin. Advan.
Human Genet., 3: 237-431, 1972.
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Aneuploid Chinese Hamster Cell Line: G-Band, C-Band, and Auto
radiographic Analyses. Chromosoma, 41: 129 144, 1973.
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Physiol.,6<5:351 409, 1965.
8. DiPaolo, J. A., Popescu, N. C., and Nelson, R. L. Chromosomal
Banding Patterns and in Vitro Transformation of Syrian Hamster
Cells. Cancer Res., 33: 3250-3258, 1973.
9. DiPaolo, J. A., Takano, K., and Popescu, N. C. Quantitation of
Chemically Induced Neoplastic Transformation of BALB/3T3
Cloned Cell Lines. Cancer Res., 32: 2686-2695, 1972.
10. Gallon, M., and Holt, S. DNA Replication Patterns of the Sex Chro
mosomes in Somatic Cells of the Syrian Hamster. Cytogenetics, 3:
97-111, 1964.
11. Hoehn, H., and Martin, G. M. Heritable Alteration of Human Con
stitutive Heterochromatin Induced by Mitomycin C. Exptl. Cell Res.,
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in Mammalian Chromosomes. Chromosoma, 34: 243 253, 1971.
13. Kato, R. Chromosome Breakage Induced by a Carcinogenic Hydro
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14. Kihlman, B. A. Molecular Mechanisms of Chromosome Breakage
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15. Lyon, M. F. Sex Chromatin and Gene Action in the Mammalian
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Fig. 1. a, karyotype of a cell with 45 chromosomes from Tumor BP45t. Quinacrine mustard stain. In addition to the marker chromosomes (M,
and Mj) and the normal fluorescent pattern of this karyotype, one of the B9 chromosomes has a fluorescent, nonbanded, translocated segment (arrow),
b, karotype of a cell with 44 chromosomes from Tumor 4NQ016t. Trypsin-Giemsa preparation. The previously described markers include M. which
is an A4 chromosome with a nonbanded, darkly stained translocation, c, metaphase of a cell from Tumor BP45t. C band preparation showing consti
tutive heterochromatin distribution of normal sex chromosomes and autosomes. In addition, C bands of the marker chromosomes M,, M 2, and M 3
are shown (arrows), d, metaphase of a cell from Tumor 4NQ0161. C band preparation showing that the translocation on the A4 chromosome is heterochromatic.
Fig. 2. a, metaphase from 4NQOI61 cell treated with thymidine-3H during early period of S phase. Majority of chromosomes covered with grains
including entire X chromosomes and M„.
b, identical metaphase after removal of emulsion, c, metaphase of a 4NQOI61 cell exposed to thymidine-3H
during a later period of S phase. Short arms of autosomes, I entire X, and one-half of the 2nd one as well as the terminal portion of M. were labeled.
d, same metaphase after removal of emulsion.
Fig. 3. Autoradiographic G and C band patterns of Markers M3, M6, and M7. Left, comparison of E20 with M3: First row, conventional stain;
second row, same chromosomes after autoradiography; third row, G band; and fourth row, C bands. Right, demonstration of the translocation to
heterochromatic arm of X to form M,; and the fusion of long arms of Chromosomes Al and A2 to form M, with I centromere. Left to right, G tech
nique, DNA replication, and C band technique.
3262
CANCER RESEARCH
VOL. 33
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Chromosome Constitutive Heterochromatin
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3264
CANCER RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.
VOL. 33
Distribution of Chromosome Constitutive Heterochromatin of
Syrian Hamster Cells Transformed by Chemical Carcinogens
J. A. DiPaolo and N. C. Popescu
Cancer Res 1973;33:3259-3264.
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