[CANCER RESEARCH 41, 2135-2140, June 1981]
0008-5472/81 /0041 -0000$02.00
Cytogenetic Evidence for Premeiotic Transformation
Cancers1
of Human Testicular
Nancy Wang,2 Kathy L. Perkins,3 David L. Bronson, and Elwin E. Fraley
Departments of Laboratory Medicine and Pathology [N. W.J, Genetics and Cell Biology [K. L P., N. W.], and Urologie Surgery [D. L. B., E. E. F.], University of
Minnesota, College of Health Sciences, Minneapolis, Minnesota 55455
ABSTRACT
To determine the point at which transformation of the germ
cell occurs during meiosis in nonseminomatous testicular can
cer, the sex chromosome compositions of 15 cell lines derived
from primary tumors or métastasesof 12 patients with testicular
cancer were analyzed by trypsin G-banding analysis and Ybody staining. The simultaneous existence of both X- and Ychromosomes in a single cell has been confirmed in 14 cell
lines. This suggests that transformation of the cell occurs
before the first meiotic division because it is known that seg
regation of X- and Y-chromosomes occurs during the first
meiotic division. An incidental finding was the presence of Barr
bodies in some cell lines containing more than one X-chromosome, which is consistent with the Known primitive nature of
testicular cancer and its ability to differentiate independently
from the male host.
INTRODUCTION
Germ cell line testicular cancers, especially teratocarcinomas, are tumors of importance to both oncologists and embryologists. By virtue of their germ cell origins and their pluripotency, they can be used as models of embryogenesis (11,
12, 14, 21 ), in the study of gene expression (1,4, 12, 13, 16,
17), and as models of neoplastic transformation (11, 22).
Despite the fact that considerable research has been devoted
to these cancers, the point at which neoplastic transformation
occurs during maturation of the germ cells is not known.
However, it is possible to determine the point of germ cell
transformation during spermatogenesis through analysis of the
sex chromosome composition of testicular tumors. If neoplastic
transformation occurs after meiotic reduction division in sper
matogenesis, then both X- and Y-chromosomes should not be
found within a single cell. Conversely, if the simultaneous
existence of the X- and Y-chromosomes is observed in the
tumor cells, it suggests a prereduction division transformation
of the germ line cells.
The sex chromosome composition of human testicular tu
mors has been studied previously by Barr body staining (7, 9,
18, 23, 27, 29), Y-fluorescence staining (2, 3), and conven
tional karyotypic analysis (8, 15). While these studies present
some evidence for the simultaneous occurrence of X- and Ychromosomes in a single cell, no precise information can be
obtained without the application of chromosome-banding tech
niques for the identification of the X- and Y-chromosomes. In
1 This work was supported by American Cancer Society Grant IN-13-R-26 and
Minnesota Medical Foundation Grant SMF-245-79.
2 To whom requests for reprints should be addressed.
3 Supported by NIH Training Grant GM-07094.
Received November 17, 1980; accepted February 17, 1981.
JUNE
the present study, trypsin G-banding analysis, Barr body anal
ysis, and Y-body analysis were performed in 15 nonsemino
matous testicular cell lines, and the sex chromosome compo
sition was analyzed in detail.
MATERIALS
AND METHODS
Cytogenetic studies were performed on 15 testicular cancer
cell lines derived from 12 patients. The cell lines have been
given the following designations: 577ML; 577MF; 577MR;
833K; 1156Q; 1218E;1242B;1255O;
1428A; 2044L; 2061H;
2102EP; 2102ER; Tera-1; and Tera-2. The origin of the cell
lines and the tissue types of the tumors from which they were
derived have been summarized previously (31 ), except cell line
1428A which was a primary tumor diagnosed as embryonal
carcinoma and yolk sac tumor and passed in culture 13 times
over 7 months.
Tera-1 and Tera-2 were established in Dr. Jörgen Fogh's
laboratory at the Sloan Kettering Institute for Cancer Research.
The rest of the cell lines were established by Bronson ef al. (5).
Cytogenetic analyses were performed on a lymphoblastoid cell
line from one of the patients, designated 577MLC, as an
internal control.
The cell lines were maintained in Roswell Park Memorial
Institute Medium 1640 containing 10 to 15% fetal calf serum
(Grand Island Biological Company, Grand Island, N. Y.), 100
units penicillin per ml, 100 ¿igstreptomycin per ml, and 10 ITIM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid buffer, and
they were subcultured weekly. Cultures were harvested for
chromosome studies when 40 to 60% confluent, treated with
0.14 /ig Colcemid per ml for 1 to 2 hr, and trypsinized to obtain
a single-cell suspension. The cells were then treated hypotonically with 0.075 M KCI or 0.7% sodium citrate for 5 to 10 min
and fixed in methanol:glacial acetic acid (3: 1, v/v). Slides
were prepared by the air-dried method and treated to obtain
trypsin G-banding by a modified method of Wang and Federoff
(30). Briefly, slides were heated on a 70°hot plate for 2 min,
treated with 0.0725% trypsin (in 0.9% NaCI solution) for 5 to
45 sec, rinsed with 0.9% NaCI solution, and stained with 2%
Giemsa in phosphate buffer for 2 to 5 min. For each cell line,
25 metaphase figures were analyzed.
To double confirm the identification of the Y-chromosome,
the Y-body staining technique of Pearson ef al. (20) was
applied on air-dried slides of the fixed tumor cells. The slides
were immersed in quinacrine hydrochloride (0.5% in deionized
water) for 5 min, rinsed gently in running tap water for 3 min,
and then mounted with 0.1 M phosphate buffer (pH 6.0). One
hundred cells were examined promptly and scored for the
presence of fluorescent Y-bodies. A Zeiss fluorescent micro
scope with BG12 or FITC exciter filter and Nos. 47 and 53
barrier filters were used.
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2135
N. Wang et al.
As a verification of the existence of an inactivated X-chromosome in some cell lines, a modification of the methods of
Darlington and LaCour (6) for Barr body staining was applied
to all 13 testicular cell lines, a male lymphoblastoid cell line
(577MLC), a female fibroblastic cell line WI38, and 2 female
amniotic cell lines. The cells were grown on either coverslips
contained in Falcon No. 3001 tissue culture dishes or on the
tissue culture dishes themselves. Four to 7 days after cultiva
tion, the coverslips or dishes were rinsed with 0.9% NaCI
solution and fixed with methanokacetic acid (3: 1, v/v) or with
100% ethanol. After fixation, the cells were hydrated from 70,
50, and 30% ethanol to distilled water and treated with 1 N HCI
at 60°for 11 min. The coverslip or culture dish was then rinsed
with distilled water for 5 min before being stained with Feulgen
stain for 60 min at room temperature. The coverslip or culture
dish was then dehydrated through distilled water and 30, 50,
70, and 95% ethanol to absolute ethanol. One hundred cells
were scored for the presence of Barr bodies.
RESULTS
Through the application of the trypsin G-banding technique
to metaphase spreads of testicular tumor cell lines, we found
that X-and Y-chromosomes were identifiable by their specific
banding patterns. The X-chromosome was identified by the
characteristic single dark band on both the long and short arms
which "frame" the centomere, and the Y-chromosome was
identified by the highly heterochromatic, darkly staining long
arms (Fig. 1). Twenty-five
trypsin G-banded metaphase
spreads per cell line were scored for the presence of X- and Ychromosomes, and the results are presented in Table 1. The
data indicate that, with the exception of the cell line Tera-1,
the X- and Y-sex chromosomes exist simultaneously in 14 of
15 cell lines.
Because of its small size, the Y-chromosome may be con
fused with other G-group chromosomes or chromosome frag
ments in metaphase spreads. Therefore, in order to confirm the
presence of the Y-chromosomes in these cell lines, we applied
the fluorescent Y-body staining technique. The technique al
lows for an evaluation of the presence of the Y-chromosome
through identification of a small, brightly fluorescent mass
located at the periphery of interphase cells which is character
istic of the intensely fluorescent heterochromatic long arms of
the Y-chromosome (Fig. 2). One hundred interphase cells per
cell line were scored for the presence or absence of the Ybody, and the results are summarized in Table 1. The Y-body
analysis confirms the results of the analysis of G-banded chro
mosomes. Fourteen of 15 cell lines contain the highly fluores
cent heterochromatic q arm of the Y-chromosome. The fre
quency of Y-bodies in Tera-1 (10%) is significantly below the
frequency of Y bodies in the control 577MLC (65%) and is thus
classified as negative for Y-bodies (Student's t test).
Through the banding analysis, we observed that 5 of 15 cell
lines contain more than one X-chromosome per diploid level of
chromosome content. Specifically, these are the cell lines
833K, 2061H, 2102EP, 2102ER, and 1428A (Table 1). In
order to investigate the plausibility that these cell lines contain
inactivated X-chromosomes, fixed cells were stained for Barr
bodies, and 200 cells/cell line were scored for the presence
of the darkly staining, peripherally located mass characteristic
of the heterochromatic X-chromosome in interphase cells (Fig.
3). Two normal human female amniotic cell lines and a human
female fibroblastic cell line (WI38) were used as the positive
controls for the Barr body staining. In addition, a lymphoblas
toid cell line, 577MLC, derived from a testicular cancer patient,
and 8 of the testicular tumor cell lines which contain only one
X-chromosome per diploid level served as negative controls for
the study.
The quantification of Barr body content in these cell lines is
summarized in Table 1. Barr bodies were observed in all 3
positive controls with a frequency of 77, 72, and 84% for the
2 amniotic cell lines and the WI38 fibroblasts, respectively. In
contrast, no Barr bodies were found in the 9 negative controls:
577MLC; 577MF; 577ML; 577MR; 1156Q; 1218E; 1255O;
1242B; and 2044L. The presence of Barr bodies exclusively in
the positive female controls confirms the reliability of the stain-
Table1
Summary of cytogenetic
studies oÃ-15 testicular tumor cell lines and 4 controls
of cells
of nor
malized'
with X- and
of cells
Y-chromo
YBarr
Barr body
with >1 X/
line577MLC577MF577ML577MR1156Q1218E1242B1255O2044LTera-1Tera-2833KE1428A2061
Cell
no.46505555541068515358665761885754464646Range42-5039-10346-9442-10545-6283-11748-10533-10247-10843-8448-10
somes100979210092100100848841008096989692%
diploid
level00812000484888459192100100100%of
bodies00002401006337745777284%
frequency2533071.682.281.35.4777284
body65675267968858633910656582574975%
H2102EP2102ERAmniotic
1Amniotic
IIFibroblast
WI38Modal
1The percentage of normalized Barr body frequency is obtained by dividing the percentage
bodies by the percentage of cells with >1 X-chromosome per diploid level.
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of Barr
CANCER
RESEARCH
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VOL. 41
Premeiotic
ing. When the same staining technique was applied to the 5
testicular tumor cell lines containing more than one X-chromosome per diploid level, a significantly high frequency of Barr
bodies was observed in 3 of them. The normalized frequencies
of Barr bodies are 82, 81, and 72% for the cell lines 2061H,
2102EP, and 1428A, respectively. The existence of Barr bod
ies in these neoplastia cells indicates that the inactivation of
the X-chromosome can occur in human testicular cancer. Fur
thermore, the absence of Barr bodies in cell lines 833K and
2102ER suggests that the sex chromosome:autosome
ratio,
perhaps, is not the only factor governing the X-chromosome
inactivation, at least in these cancers.
DISCUSSION
In a previous study, we reported the hyperdiploid to hypotriploid nature of 14 nonseminomatous human testicular cancers
which suggests a diploid cell origin of this neoplasia (31). In
this study, we demonstrated that the histogenesis of nonsemi
nomatous testicular germ cell tumors can be resolved through
the analysis of their sex chromosome composition. During
meiosis, the X- and Y-chromosomes, like each pair of homol
ogous autosomes, are separated at the first meiotic division
(Chart 1). Thus, if neoplastic transformation occurs after the
first meiotic division (i.e., if the cells of origin are haploid), Xand Y-chromosomes should not be found within a single cell.
In contrast, simultaneous existence of X- and Y-chromosomes
in all of the tumor cells indicates that neoplastic transformation
occurs before reduction division (i.e., the cells of origin are
diploid).
Through trypsin G-banding analysis of nonseminomatous
human testicular tumors and through Y-body analysis of the
interphase cells, we observed simultaneous existence of Xand Y-chromosomes in the cells of 14 of 15 tumors. These
results, along with the hyperdiploid to hypotriploid nature, are
most compatible with a diploid cell origin for these tumors. Our
findings are substantiated by the observation of Atkins (3) of
both Y-bodies and Barr bodies in 2 human testicular teratomas.
Stevens' (25, 26) finding that murine testicular teratomas orig
inate in diploid primordial germ cells also supports our conclu
sion.
While a diploid cell origin is the simplest explanation of our
XY
First
Meiotic
Division
Second Meiotic
Chart 1. Diagram of the segregation
matogenesis.
JUNE
of X- and Y-chromosomes
Division
during sper-
Transformation
of Human Testicular Cancers
results, there are other possibilities, most notably autofertilization or fusion of haploid cells (7-9, 28, 29). However, the
expected sex chromosome composition of tumors derived
through random fusion of haploid cells is inconsistent with our
observation that X- and Y-chromosomes exist simultaneously
in 14 of 15 testicular tumors.
The sex chromosome content of the cell line Tera-1 (one Xchromosome, no Y-chromosome) fits most closely the ex
pected sex chromosome content of a tumor of haploid cell
origin. However, in view of our results for the other 14 cell
lines, it is also possible that this tumor arose from a diploid
germinal cell with subsequent loss of the Y-chromosome. Aneuploidy in tumor cells is well documented; specifically, there
are reports of male tumor cells without Y-chromosomes (2,
24).
It is known that the sex chromatin composition of testicular
tumors may differ from that of the male host from which it arises
(7-9, 18). However, the significance of this fact has not been
explored. According to the hypothesis of Lyon (10), X-chro
mosome inactivation is a process of differentiation which oc
curs only in embryonic cells, and the status of the X-chromo
some does not alter once heterochromatinization
occurs (19).
In this study, we identified 3 cell lines which contain Barr
bodies. Each of these cell lines contains more than one Xchromosome per diploid chromosomal level. The presence of
Barr bodies in some testicular tumors indicates that they have
differentiated independently of the karyotypically normal male
host. Furthermore, since X-chromosome inactivation is a proc
ess which occurs during embryogenesis, these findings reiter
ate the known primitive nature of some testicular tumor tissue.
Barr bodies were observed only in cell lines which contain
more than one X-chromosome per diploid chromosome level,
as predicted by the equation:
No. of Barr bodies = No. of X-chromosomes
-
ploidy no.
However, 2 cell lines which contain more than one X-chromo
some per diploid level (2102ER and 833KE) do not contain
Barr bodies. Apparently, the ratio of the number of X-chromo
somes to autosomes is not the only factor which influences Xchromosome inactivation in these cell lines. Particularly inter
esting is a comparison of the results for the cell lines 2102EP
and 2102ER, which are, respectively, derived from a primary
tumor and retroperitoneal metastasis of the same patient. The
primary, tumor cells contain Barr bodies in this case while the
metastatic tumor cells do not, although both of them are of
almost identical chromosome composition (31 ). While culturing
artifacts cannot be eliminated as an explanation for our results,
a study by Myers (18), in which he observed Barr bodies only
in certain histologically separate regions of thin-sectioned hu
man tumors, suggests that the presence or absence of Barr
bodies may reflect differences in degree of differentiation of
the neoplastic tissue. This heterogeneity with respect to the
potential of X-chromosome inactivation in the primary tumors
may be of importance in metastatic selection.
To extend the observation reported herein, studies of the
Barr body frequencies should be correlated with the histopathology and degree of malignancy of the neoplasia. Further
more, those cell lines identified with more than one X-chro
mosome per diploid level may be used to elucidate the nature
of X-chromosome inactivation for human cells in vitro. With
1981
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2137
N. Wang et al.
regard to the analysis of sex chromosome composition, further
studies should be performed on primary tumors, both seminomas and nonseminomas, to verify the histological
these 2 different types of germ cell line neoplasia.
origin of
14.
15.
ACKNOWLEDGMENTS
16.
We thank Beth Trend and Hannelore D. Asmussen for their technical help and
Diane Konzen tor the typing and critical reading of the manuscript. Tera-1 and
Tera-2 cells were a gift from Dr. JörgenFogh, Sloan-Kettering Institute for Cancer
Research, New York, N. Y.
17.
REFERENCES
19.
1. Artzt, K., Bennett, D., and Jacob, F. Primitive terato carcinoma cells express
a differentiation antigen specified by a gene at the T-locus in the mouse.
Proc. Nati. Acad. Sei. U. S. A., 71: 811-814, 1974.
2. Atkin, N. B. Y bodies and similar fluorescent chromocentres in human tumors
including teratoma. Br. J. Cancer, 27: 183-189, 1973.
3. Atkin, N. B. Y chromosomes and quinacrine fluorescence technique. Br.
Med. J., 4: 118, 1970.
4. Bernstine, E. G., Hooper, M. L., Grandchamp, S., and Ephrussi, B. Alkaline
phosphatase activity in mouse teratoma. Proc. Nati. Acad. Sei. U. S. A., 70:
3899-3903,
1973.
5. Bronson, D. L., Andrews, P. W., Solter, D., Cervenka, J., Lange, P. H., and
Fraley, E. E. Cell line derived from a metastasis of a human testicular germ
cell tumor. Cancer Res., 30: 2500-2506,
1980.
6. Darlington, C., and LaCour, L. The Handling of Chromosomes. London: Allen
and Unwin, 1960.
7. Dayan, A. D. Nuclear sex of testicular teratomas. Br. J. Cancer, 17: 46-49,
1963.
8. Gallon, M., Benirschke, K., Baker, M. C., and Atkin, N. B. Chromosomes of
testicular teratomas. Cytogenetics (Basel), 5. 261-275, 1966.
9. Hunter, W. F., and Lennox, B. The sex of teratoma. Lancet, 267: 633-634,
1954.
10. Lyon, M. F. Possible mechanisms of X chromosome-inactivation.
Nat. New
Biol., 232: 229-232, 1971.
11. Martin, G. R. Teratocarcinomas as a model system for the study of embryogenesis and neoplasia. Cell, 5. 229-243, 1975.
12. Martin, G. R. Teratocarcinomas
and mammalian embryogenesis. Science
(Wash., D.C.), 209: 768-776, 1980.
13. Martin, G. R., Epstein, C. J., Travis, B., Tucker, G., Yatziv, S., Martin, D.,
20.
2138
18.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Clift, S., and Cohen. S. X-chromosome ¡nactivation during differentiation of
female teratocarcinomas stem cells in vitro. Nature (Lond.), 27): 329-333,
1978.
Martin, G. R., Wiley, L. M., and Damjanov, I. The development of cystic
embryoid bodies in vitro from clonal teratocarcinoma stem cells. Dev. Biol.,
61: 230-244, 1977.
Martineau, M. Chromosomes in human testicular tumors. J. Pathol., 99:
271-282, 1969.
McBurney, M. W., and Adamson, E. D. Studies on the activity of the Xchromosome in female teratocarcinoma
cells in culture. Cell, 9: 57-70,
1976.
Monk, M. Biochemical studies on mammalian X-chromosome activity. Dev.
Mammals, 3: 189-223, 1978.
Myers, L. Sex chromatin in teratomas. J. Pathol. Bacteriol., 78: 43-55,
1959.
Ohno, S. X-chromosome behavior and aneuploidy. Proc. Am. Assoc. Cancer
Res., 3. 349, 1962.
Pearson, P., and Bobrow, M. Technique for identifying Y chromosomes in
human ¡nterphase nuclei. Nature (Lond.), 226. 78-80, 1970.
Pierce, G. B., Dixon, F. J., and Verney, E. L. Teratocarcinogenic
and tissue
forming potentials of the cell types comprising neoplastic embryoid bodies.
Lab. Invest., 9: 583-602, 1960.
Pierce, G. R. Teratocarcinoma:
model for a developmental concept of
cancer. Curr. Top. Dev. Biol., 2: 223-246, 1967.
Pierce, G. R., and Nakane, P. D. Nuclear sex of testicular teratomas of
infants. Nature (Lond.), 274: 820-821, 1967.
Sellyai, M., and Vass, L. Absence of fluorescent Y bodies in some malignant
epithelial tumours in human males. Preliminary results. Eur. J. Cancer, 8:
557-559, 1972.
Stevens, L. C. Testicular teratomas in fetal mice. J. Nati. Cancer Inst., 28
247-268, 1962.
Stevens, L. C. Origin of testicular teratomas as from primordial germ cells in
mice. J. Nati. Cancer Inst., 38: 549-552, 1967.
Tavares, A. S. On the sex of cancer and teratomata cells. Lancet, Õ:948949, 1955.
Teplitz, R. L., and Beutler, E. Mosaicism, chimerism and sex chromosome
inactivation. Blood, 27: 258-271, 1966.
Theiss, E. A., Ashley, D. J. B., and Mostofi, F. K. Nuclear sex of testicular
tumors and some related ovarian and extragonadal neoplasms. Cancer
(Phila.), 13: 323-327, 1960.
30. Wang, H. C., and Federoff, S. Banding in human chromosomes treated with
trypsin. Nat. New Biol., 235. 52-54, 1972.
31. Wang, N., Trend, B., Bronson, D. L., and Fraley, E. E. Non-random abnor
malities in chromosome 1 in human testicular cancers. Cancer Res., 40:
796-802, 1980.
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VOL.
41
Premeiotic
Transformation
of Human Testicular Cancers
I
\
Fig. 1. A metaphase spread from a testicular cancer cell line showing 2 X-chromosomes and 1 Y-chromosome.
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1981
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2139
N. Wang et al.
Fig. 2. Y-body in a testicular cancer
cell line visualized by fluorescence micros
copy (arrow).
\
Fig. 3. Barr body of a testicular cancer cell line visualized by light microscopy
(arrow).
2140
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VOL. 41
Cytogenetic Evidence for Premeiotic Transformation of Human
Testicular Cancers
Nancy Wang, Kathy L. Perkins, David L. Bronson, et al.
Cancer Res 1981;41:2135-2140.
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