J. Cell Sri. 56, 113-130 (1982)
Printed in Great Britain © Company of Biologists Limited 1982
THE ANALYSIS OF MALIGNANCY
BY CELL FUSION
IX.* RE-EXAMINATION AND CLARIFICATION
OF THE CYTOGENETIC PROBLEM
E. P. EVANS, M. D. BURTENSHAW, B. B. BROWN,
R. HENNION AND H. HARRIS
Sir William Dunn School of Pathology, University of Oxford,
Oxford OXi 3RE, England
SUMMARY
Previous experiments with crosses between malignant and diploid mouse cells had shown
that the reappearance of malignancy in hybrids in which it was initially suppressed was
associated in some cases with the elimination of the chromosomes 4 derived from the diploid
parent cell. In others, however, this did not appear to be so. In the present study, we have
re-examined the role of the diploid chromosomes 4 in the suppression of malignancy using
natural polymorphisms of the centromeric heterochromatin to identify the parental origin
of the chromosomes 4 in the hybrid cells. We now find that the diploid chromosomes 4 are
indeed involved in the suppression of malignancy in all the tumours that we have examined,
which include a carcinoma, a melanoma, a sarcoma and a lymphoma. In all crosses between
these malignant tumour cells and diploid fibroblasts, there is selective pressure in vivo against
the chromosomes 4 derived from the diploid cell and in favour of the chromosomes 4 derived
from the malignant cell. This indicates that the chromosomes 4 in all these tumours are in
some way functionally different from the chromosomes 4 of the diploid fibroblast. Reappearance
of malignancy in hybrids in which it was initially suppressed may result from a reduction in
the number of diploid chromosomes 4, an increase in the number of malignant chromosomes
4, or both. The gene on the diploid chromosome 4 responsible for the suppression of malignancy
acts in a dose-dependent manner.
INTRODUCTION
Since the original observation of Harris, Miller, Klein, Worst & Tachibana (1969)
numerous authors have reported that the fusion of malignant cells with normal
cells may generate hybrids in which malignancy is suppressed. (For a recent review,
see Harris (1981).) A good deal of effort has been expended on the cytogenetic
analysis of such hybrids, both intraspecific and interspecific, and although it is
clear that the reappearance of malignancy in hybrids in which it was initially suppressed is associated with the elimination of specific chromosomes, the precise
karyological basis for the suppression of malignancy has still not been satisfactorily
resolved. The most detailed study in intraspecific crosses, which, because of their
relative chromosomal stability, have some advantages for this kind of analysis, is
that of Jonasson, Povey & Harris (1977). These authors found that in crosses
• Paper VIII in this series is by Jonasson & Harris (1977).
ii4
E. P. Evans, M. D. Burtenshaw, B. B. Brown, R. Hennion and H. Harris
between two different kinds of malignant mouse cells and diploid mouse fibroblasts,
there was selective pressure both in vitro and in vivo against the chromosomes
number 4 derived from the normal parental cell. This finding obviously indicated
that in these two tumours, a melanoma and a sarcoma, the chromosomes 4 derived
from the tumour cell differed in some way from their normal homologues. However,
when crosses between the normal fibroblasts and other mouse tumour cells were
examined, selective pressure against the normal chromosomes 4 was not obvious.
Moreover, in interspecific crosses between a malignant mouse melanoma and a
human diploid cell, some hybrids were isolated in which malignancy was suppressed
but in which no human chromosomes were seen (Jonasson & Harris, 1977). This
raised the possibility that extrachromosomal factors might be involved in the suppression of malignancy; but Rodgers (1979), reinvestigating these hybrids with nucleic
acid hybridization techniques, showed that, although no human chromosomes were
visible, these hybrids did contain a small amount of human DNA. The case for
extrachromosomal factors in the suppression of malignancy was thus greatly weakened,
although there have been reports of such suppression in 'cybrids' made by fusing
malignant cells with enucleated non-malignant cells (Howell & Sager, 1978; Giguere
& Morais, 1981).
The refinement of cytogenetic techniques that has taken place since the paper by
Jonasson, Povey & Harris (1977) was published prompted us to reinvestigate the
role of chromosome 4 in regulating the malignancy of intraspecific mouse hybrids.
It was clearly of the greatest importance to determine whether, indeed, a single
chromosome was involved in this regulation in a range of different tumours, or
whether the two instances reported by Jonasson et al. were both special cases. The
technique we have adopted in the present work for identifying the parental origin
of the chromosomes number 4 in the hybrid cells differed in one important particular
from that used by Jonasson et al. In the latter experiments, identification was made
possible by the use of fibroblasts explanted from mice bearing specific chromosome
translocations. However, some recent observations we have made led us to suspect
that the ease with which certain chromosomes were eliminated from hybrids might
be influenced by whether they were present in translocations or not. Clearly, if
certain translocated chromosomes are selectively retained or selectively eliminated,
this could introduce a serious bias in the results that would be obtained when cells
of different kinds are fused together. For this reason we chose in the present
experiments to exploit a natural polymorphism in the structure of chromosome 4
that is found in different strains of mice. This polymorphism is clear enough to
permit unequivocal identification of the parental origins of the chromosomes 4 in
appropriately constructed hybrid cells.
MATERIALS AND METHODS
Parental and hybrid cell lines
All the malignant cells used in the present study are transplantable tumours of the mouse.
PG19 is a melanoma, YACIR IMP" a lymphoma, AoHT and SEWA sarcomas and TA3HaB
a mammary carcinoma. They have been described in detail in previous papers of this series
Analysis of malignancy. IX
115
(Harris, 1971; Wiener, Klein & Harris, 1973, 19746; Jonasson et al. 1977). The malignant
cells were fused by means of u.v.-irradiated Sendai virus (Harris & Watkins, 1965) with
fibroblasts explanted from A/Sn, CBA/H-T,T, and Rb(i6.i7)7Bnr mice. Details concerning
these mouse strains, and the techniques used for isolating hybrid clones from the fused cell
populations are given by Jonasson et al. (1977).
Hybrids containing one set of A9HT chromosomes and two sets of CBA/H-T,T, fibroblast chromosomes were obtained by fusing A9HT cells with fibroblasts that had been exposed
to cytochalasin B. The fibroblasts were grown for 48 h in medium containing cytochalasin B
at a concentration of 10 /ig/ml and then for a further 3 h in medium without cytochalasin B.
After this treatment, between 30 and 40 % of the cells were found to be tetraploid. Fibroblast
cultures containing a high proportion of tetraploid cells were fused with A9HT cells, and
hybrid clones were isolated in the usual way. Cytogenetic analysis of these clones permitted
hybrids between A9HT cells and tetraploid fibroblasts to be selected.
The tumorigenicity of the hybrid cells was assessed by subcutaneous inoculation of cell
suspensions into sublethally irradiated, syngeneic new-born mice, as described in previous
publications.
Cytogenetic techniques
Cells in tissue culture were pretreated with colcemid at a concentration of 01 /tg/ml for
up to 1 h. Attached cells were dislodged by shaking. The cell suspensions were subjected
to a hypotonic treatment of 0-56 % (w/v) KC1 for 8 min. They were fixed initially as a pellet
in three changes of 3 parts methanol to 1 of glacial acetic acid, resuspended and given a final
change of fixative. Spreads were air-dried onto dry slides.
The slides were aged for between 3 and 10 days at room temperature then G-banded by a
modification of the combined ASG/trypsin method described by Gallimore & Richardson
(1973). No attempt was made to analyse cells before photographing them: the cells were
selected on the basis of their having a chromosome count approximating to that expected
and of their being free of chromatid aberrations and an excessive number of fragments. At
least five such cells were photographed on Kodak Technical Pan 2415 film. Some of the
prints made were analysed either- by cutting out the chromosomes and, wherever possible,
arranging them into homologous groups, or by numbering the chromosomes on the prints
with a felt-tipped pen. All grouping and numbering followed the system recommended by the
Committee on Standardized Genetic Nomenclature for Mice (Committee, 1972).*
Because of the presence of many metacentrics in A9HT and its hybrid clones, the total
chromosome number was expressed as the number of centromeres present. In all the other
hybrid clones and the tumours derived from them, chromosome number was expressed as
the total number of chromosome arms or NF.
Few morphological differences exist between the homologous chromosomes of different
mouse strains, and, in many cases, this is also true for the tumours derived from the mice.
The most marked and consistent polymorphisms are found in the C-bands (centromeric
heterochromatin). The Committee on Standardized Genetic Nomenclature for Mice recommends the designation He for centromeric heterochromatin coupled to the chromosome
number on which it lies. (Thus HC4 = centromeric heterochromatin on chromosome 4.)
Differences in the size of the He have been designated He" (normal), He1 (large) and He'
(small). Wherever possible, we have made use of C-band differences to identify the parental
origin of the chromosomes 4 in the hybrid clones (Fig. 1). PG19 is homozygous for HC4' and
was therefore fused with A/Sn fibroblasts, which are Hc4". Both YACIR IMP" and Ta3HaB
are Hc4* and were therefore fused with CBA/H-T,T, fibroblasts, which are HC41. In the
case of A9HT and SEW A there was no need to use markers of this kind since their chromosomes
4 are already marked by translocations, in the former by centric fusions (4-4 and 4-15) and
in the latter by a partial tandem translocation that produces a long chromosome made up of
chromosome 4 material from band Ai to C6 and then from C2 to E2 (Committee, 1974). This
chromosome therefore represents much of two copies of chromosome 4 and, as there is no other
identifiable chromosome 4 present, it may have evolved from the original homologous pair.
• The tables given in the present paper provide summaries of the cytogenetic studies.
Full details can be made available on request.
n6
E. P. Evans, M. D. Burtenshaw, B. B. Brown, R. Hennion and H. Harris
PG19
A/Sn
YACIR
CBAT 6 T 6
It i. K I)
A9HT
C B A T6 1J 6
Ta3Ha
SEWA
Rb7Bnr
CBATJ"
6 •6
If M
Fig. i. The chromosome 4 markers used to identify the parental origin of the
chromosomes 4 in hybrid cells. PG19 is homozygous for HC41, A/Sn homozygous
for Hc4'- YACIR IMP- is homozygous for Hc4', CBA/H-T.T, homozygous for
HC41. In A9HT the chromosomes 4 are involved in a 4-4 and 34-15 centric fusion,
CBA/H-T,T, has normal chromosomes 4. In SEWA the only chromosome identifiable as a 4 is in the form of a partial tandem translocation, Rb7Bnr chromosomes 4
are normal. Ta3HaB is homozygous for Hc4", CBA/H-T,T, homozygous for HC41.
RESULTS
Hybrids in which malignancy is stably suppressed
PGig melanoma x A/Sn fibroblast hybrids. It has previously been shown that in
hybrids between PG19 and diploid fibroblasts, there is selective pressure, both
in vitro and in vivo, against the chromosomes 4 derived from the diploid parent
cell. Hybrids in which both diploid chromosomes 4 were retained grew very slowly
and could not be cloned. A fresh set of hybrids was isolated between PG19 and
fibroblasts derived from the A/Sn mouse, in which the chromosomes 4 are distin-
Analysis of malignancy. IX
117
Table 1. Chromosome 4 constitution of PGiq x A/Sn clone 14
Copies
of chromosome 4
per cell
PG19
A/Sn
Expected in complete bi-parental hybrid
2
Observed (mean of 10 cells)
2
• Total number of chromosome arms.
NF*
81
80s
Table 2. Growth of cell lines in vivo
Take incidence
(no. tumours/no.
animals injected)
Mean
latent period
(days)
5 x 10*
5 x 10*
5 x 10*
7/7
0/22
0/10
6
—
—
5 x 10'
YACIR IMPYACIR IMP" x CBA/H-T.T, fibroblast hybrid
10/10
12
10'
10'
io»
10'
io»
18/20
0/8
5°
—
Cell line
PG19
PG19 x A/Sn fibroblast hybrid
Clone 14
Clone
Clone
Clone
Clone
Clone
1G1
1G8
iG8a
iG8b
iG8c
A9HT
A9HT x CBA/H-T.T, fibroblast hybrid
Clone 2
Clone 4
Clone 9
Clone 14
No. cells
injected
5x
5x
5x
5x
5x
5/6
5/5
25
28
9/9
3°
5 x 10*
9/9
19
5 x 10*
5 x 10*
5 x 10*
5 x 10*
11/12
16/16
33
5/12
14/19
25
29
20
1 A9HT x 2 CBA/H-T.T, fibroblast hybrid
Clone 2
Clone 3
Clone 9
5 x 10*
5 x 10*
5 x io 4
5 x 10'
SEWA
SEWA x Rb(i6- i7)7Bnr fibroblast hybrid
5 x io 4
Clone F when first examined
5 x io 6
after cultivation
5 x 10'
Clone D
5 x 10*
TA3 HaB
TA3 HaB x CBA/H-T 6 T 0 fibroblast hybrid
Clone Ci
Clone F2
Clone B2
5 x 10*
5 x 10*
5 x 10*
s/s
4/7
5/i3
27
3O
53
6/6
10
i/9
27
6/6
4/4
10
13/13
10
7/7
6/6
12
14/14
7
11
13
n8
E. P. Evans, M. D. Burtenshaw, B. B. Brown, R. Hennion and H. Harris
UK Hii (»> HU !
t
m
1
?mi im iiii mi j»»
1
Mil
11
Iiti
It
nil
16
III
17
9
10
I4I
HI! « <
13
14
18
19
15
»
X Y
Fig. 2. Karyotype of a cell from PG19 melanoma x A/Sn diploid fibroblast hybrid
clone 14. Of the 81 chromosomes to be expected from the fusion of these two parent
cells, 80 are retained in this hybrid. (In PG19 a number of chromosomes are translocated, as described by Jonasson et al. 1977.) The He* chromosomes 4 derived
from the A/Sn fibroblast (the pair on the left of the group of four) are clearly
distinguishable from the He1 chromosomes 4 derived from PG19 (the pair on the
right of the group of four).
guishable from those of the PG19 cell by the centromeric heterochromatin polymorphism described above. Despite its slow growth, we succeeded on this occasion
in isolating one clonal line (clone 14) in which both diploid chromosomes 4 were
retained (Table 1). It will be seen that these hybrids have retained something very
close to the chromosome constitution one would expect from the fusion of the two
parent cells. Fig. 2 shows the karyotype of a typical cell. The malignant and the
diploid chromosomes 4 are clearly distinguishable. Clone 14 produced no tumours
in 22 mice with inocula of 5 x io 4 cells per mouse and none in 10 mice with inocula
of 5 x io5 cells per mouse (Table 2). It is clear that suppression of malignancy in
Analysis of malignancy. IX
119
this clone is extremely stable. Very large numbers of animals will need to be
inoculated with these hybrids if tumours are to be obtained for analysis.
YACIR IMP- lymphomaxCBA/H-TtTz fibroblast hybrids. The origin and
characteristics of clone iG, a hybrid clone derived from this cross, have been
previously described (Jonasson et al. 1977). The clone initially produced no tumours
in 12 mice with inocula of 4X io 8 cells per mouse, but, after several weeks' cultivation in vitro, a malignant sub-population was generated that overgrew the cultures.
Early passages of clone iG were re-cloned, and the secondary clones tested for
Table 3. Chromosome 4 constitution of YACIR IMP~ x CBA/H-TeTe
clone 1G8
Copies
of chromosome 4
per cell
YACIR
Expected in complete bi-parental hybrid
When first examined (mean of 6 cells)
After 6 weeks growth in vitro
(mean of 35 cells)
CBA/HT,T,
2
2
1
1
YACIR(Df)#
1
1
1-9
NF
80
76-3
76-9
• YACIR(Df), Df, nomenclature rule for deficiency/deletion. Deleted from band C6 to
E2.
Table 4. Chromosome 4 constitution of YACIR IMP~ x
clone 1G1 and tumour
CBA/H-TtTe
Copies
of chromosome 4
CBA/H-
YACIR
Expected in complete bi-parental hybrid
When first examined (mean of 7 cells)
Tumour 1 (mean of 10 cells)
2
1-7
2
T.T,
NF
2
1
1
80
75'6
68-7
tumorigenicity. Two of these secondary clones were selected for further study.
Clone 1G8 produced no tumours with inocula of 5 x io 6 cells per mouse; clone 1G1
produced tumours in about 90% of the animals with this inoculum (Table 2).
When first examined and assayed for tumorigenicity, the great majority of the cells
in clone 1G8 contained only one chromosome 4 from the YACIR IMP" parent
cell and one from the CBA/H-T e T 6 fibroblast. Six weeks later, the cultures had
been overgrown by cells containing two chromosomes 4 from the YACIR IMP"
parent cell, one bearing a small deletion, and two chromosomes 4 from the fibroblast
parent cell (Table 3). This illustrates an observation made previously by Jonasson
et al. (1977), namely, that hybrid cells containing only two chromosomes 4 (the
equivalent, in principle, of monosomy for chromosome 4 in a diploid cell) are at
120
E. P. Evans, M. D. Burtenshaw, B. B. Brovm, R. Hennion and H. Harris
a selective disadvantage and tend to be overgrown both in vitro and in vivo by cells
in which one or more of the residual chromosomes 4 have been duplicated. The
clone 1G8 cells containing two chromosomes 4 from each parent cell still failed to
produce tumours with inocula of 5 x io 5 cells per animal (Table 2). On the other
hand, the tumorigenic clone 1G1 contained both chromosomes 4 from the YACIR
IMP" parent cell, but only one chromosome 4 from the diploid parent cell. No
systematic differences between clone 1G1 and clone 1G8 were seen for any other
chromosome. The cytogenetic analysis of this clone and of a tumour derived from
it are shown in Table 4.
The difference in tumorigenicity between clone 1G8 and clone 1G1 suggested
that if the chromosomes 4 derived from the diploid parent cell were involved in the
suppression of malignancy, the loss of one of these chromosomes was enough to
produce a marked increase in tumorigenicity. This idea was tested by a further
Table 5. Chromosome 4 constitution of subclones of YACIR IMP~ x
clone 1G8 selected in agarose
CBA/H-TaTt
Copies of chromosome 4 per cell
YACIR
Clone A (mean of 20 cells)
Clone B (mean of 6 cells)
YACIR
(DO
CBA/HT,T,
NF
0-9
1
1
I-I
73-7
o-8
1-2
73-3
YACIR(Dp)*
1
I-I
1
ro
76-3
Clone C (mean of 10 cells)
• YACIR(Dp), Dp, nomenclature rule for duplication. A complex duplication resulting in
a long chromosome that in sequence consists of band Ai to C6 and then of A2 to E2.
examination of clone 1G8. Since no tumours were produced by direct inoculation
of clone iG8, an effort was made to select from this clone subclones capable of
growth in agarose, in the hope that such subclones might be enriched for cells
capable of progressive growth in vivo. From three dishes, each seeded with io 5 cells
in agarose, as described by Steinberg & Pollack (1979), five primary colonies were
obtained, of which three survived isolation and further subculture (clones iG8a,
b, c). These gave between 80 and 100% take incidences with inocula of 5 x 10s cells
per mouse (Table 2). The cytogenetic analysis of these subclones is shown in Table 5.
All three clones showed the loss of one chromosome 4 derived from the diploid
fibroblast, and one clone now had three copies of the chromosome 4 derived from
the malignant parent cell. The tumours produced by these two clones showed no
further systematic changes in chromosome constitution. It thus appears that, in this
cross, the loss of a single diploid chromosome 4, provided that both malignant
chromosomes 4 are retained, is enough to allow the malignant phenotype to reappear.
The gene on chromosome 4 responsible for the suppression of malignancy thus
appears to act in a dose-dependent fashion.
Analysis of malignancy. IX
121
Hybrids in which the suppression of malignancy is unstable
AgHTx CBA/H-T6T6 fibroblast hybrids. We have previously shown that hybrids
between diploid cells and the A9HT sarcoma give variable take incidences (Wiener,
Klein & Harris, 1974 a). Some clones produce few tumours, others at the same
inoculum give high take incidences. On continued cultivation in vitro, those clones
in which malignancy was initially suppressed generate tumorigenic subpopulations
that progressively overgrow the cultures. We have re-examined the behaviour of
the chromosomes 4 in a fresh set of A9HT x CBA/H-T 6 T 9 fibroblast hybrids. A
detailed examination of the karyotype of A9HT itself revealed that this malignant
cell line contains three chromosomes 4, one in a Robertsonian translocation with
Table 6. Chromosome 4 constitution of AgHTx CBA/H-T6Te clones 2, 4, 9 and 14
when first examined and after several weeks' cultivation in vitro
Copies of chromosome 4 per cell
A9HT
4-4
Expected in complete bi-parental
hybrid
Clone 2 (mean of 5 cells)
Clone 2 after cultivation
(mean of 10 cells)
Clone 4 (mean of 5 cells)
Clone 4 after cultivation
(mean of 5 cells)
Clone 9 (mean of 6 cells)
Clone 9 after cultivation
(mean of 7 cells)
Clone 14 (mean of 5 cells)
Clone 14 after cultivation
(mean of 9 cells)
4-15
Total
CBA/H- Total no. of
T,T, chromosomes
1
3
2
92
1
1
09
3
29
2
1
1-9
908
931
1
1 + /i cell\ 3 2
2
904
1
I 49 /
1
1
3
i-6
896
1
1
1
3
3
2
1
907
92
19
1
1
3
2
932
1
I-I
3-i
17
92-4
chromosome 15 and two in a Robertsonian 4-4 translocation. Complete bi-parental
crosses between A9HT and a diploid fibroblast thus differed from the PG19 and
the YACIR IMP" crosses described above in having ab initio a greater complement
of chromosomes 4 derived from the malignant parent cell than from the diploid
parent cell. A number of clones containing a complete set of chromosomes 4 from
both parent cells were isolated and examined. Their take incidences are given in
Table 2. It will be seen that, compared with the parental A9HT cell, these hybrid
clones showed either a reduced take incidence or a longer latent period before the
appearance of the tumours, or both. However, the reduction in tumorigenicity is
far less impressive than that obtained with the PG19 and YACIR IMP~x fibroblast
crosses. This difference might have resulted from the more rapid elimination of the
5
CEL56
122 E. P. Evans, M. D. Burtenshaw, B. B. Brown, R. Hennion and H. Harris
diploid chromosomes 4 in this cross, but, as shown in Table 6, both diploid chromosomes 4 are largely retained in these hybrids for several weeks of cultivation in vitro.
It seems more probable, given the indication that the diploid chromosome 4 acts
to suppress malignancy in a dose-dependent fashion, that an increased dosage of
the malignant chromosome 4 tends to counteract this suppression. All tumours
produced by the A9HT x fibroblast hybrids have eliminated either one or both of
the chromosomes 4 derived from the diploid parent cell (Table 7).
The interaction between the diploid and the malignant chromosomes 4 was
examined further in hybrids between A9HT cells and tetraploid CBA/H-T 6 T 8
fibroblasts. Several hybrid clones containing one A9HT chromosome set and two
fibroblast chromosome sets were isolated, but none of them had retained the full
complement of diploid chromosomes 4. Three such clones, 2, 3 and 9, were studied
Table 7. Chromosome 4 constitution of the tumours derived from
AgHTx CBA/H-T6T6 clones 2, 4, 9 and 14
Copies of chromosome 4 per cell
A
A 9 HT
A
.-,.
(Ti (mean of 5 cells)
Clone 4 {rr, )
,, N
r
^ \ T 2 (mean of 10 cells)
.-,.
(Ti
(mean of D5 cells)
.'
Clone
4 L
, (mean
\12
ofr 10 cells)
Clone 9 T2 (mean of 6 cells)
-,.
f T i (mean of 6 cells)
, .. :
Clone 14 ( „ ;
^ \T2 (mean ofr 6 cells)
4'4
4-15
Total
1
1
I
CBA/H- Total no. of
T.T, chromosomes
o-8
1
I
1
3
3
3
3
1
1
3
03
1
I
1
17
3
3-7
0
0
1
1
07
1
07
87-8
88-3
87-3
857
88-s
8i-8
80-5
in detail. As shown in Table 8, all of them, as soon as they could be analysed, had
already eliminated two or more of the chromosomes 4 derived from the tetraploid
CBA/H-T 6 T 6 fibroblast, and two of them showed an increase in the number of
chromosomes 4 derived from the A9HT parent cell. It is thus clear that in these
hybrids selection against the diploid chromosomes 4 and in favour of the malignant
chromosomes 4 is already operative in vitro. As shown in Table 2, the three hybrid
clones gave variable take incidences, and the latent periods preceding the appearance
of tumours were again prolonged compared with A9HT. The cytogenetic analysis
of the tumours produced from these hybrids is shown in Table 8. It is clear that
there is further selection against the diploid chromosomes 4 and in favour of the
malignant chromosomes 4 in vivo. Compared with the cells injected, all tumours
show a further increase in the number of malignant chromosomes 4 and a further
decrease in the number of diploid chromosomes 4. In some of the tumours, the
latter are eliminated altogether. These findings thus lend strong support to the
view that the suppression of malignancy in hybrids between malignant and diploid
Analysis of malignancy. IX
123
Table 8. Chromosome 4 constitution ofAgHTx tetraploid CBA/H-TeT9
clones 2, 3 and 9 and tumours derived from them
Copies of chromosome 4 per cell
A9HT
K
44
4-15
Total
CBA/H- Total no. of
chromosomes
T.T 8
Expected in complete bi-parental
hybrid
1
I
3
4
132
(mean of 20
T i (mean of 10
Clone 2 •
T2 (mean of 10
T3 (mean of 10
1-25
1-7
i-4
17
42
5-4
44
5'5
I-IS
2-O
0
0
O-2
i3S
1217
113-1
m-6
cells
cells)
cells)
cells)
f
(mean of 10 cells)
Clone 3 J. T i (mean of 10 cells)
[T2 (mean of 10 cells)
(
(mean of 10 cells)
n ,
Clone 9 •[„ ;
„ (
IT2 (mean ofr 10 cells)
2-O
i-6
IS
I-O
19
i-o
I-I
2-1
2-O
I-O
I-O
i-8
29
3-9
41
42
°-5
1199
1043
107-1
3-0
6-S
2-O
O
114-0
II2-O
23
0-4
cells involves a dose-dependent interaction between the malignant and the diploid
chromosomes 4.
SEW A x Rb{i6.ij)7Bnrfibroblast hybrids
Crosses between SEWA and diploid fibroblasts, like the corresponding A9HT
crosses, show a reduction in tumorigenicity compared with that of the parental
SEWA cells (Wiener, Klein & Harris, 1971), but this is again less pronounced and
less stable than that seen with PG19 and YACIR IMP" x fibroblast crosses. SEWA
is an unpromising cell for detailed cytogenetic analysis. Only one chromosome is
identifiable as a derivative of chromosome 4 and, since it takes the form of a partial
tandem translocation, it represents approximately two copies of this chromosome.
It may have evolved either from the original homologous pair or by a complex
duplication of a single chromosome. Other chromosomes 4 may be present, but
they cannot be recognized with confidence in the multiple rearrangements that
characterize this cell. We therefore do not know with any certainty, in this cross,
how many equivalents of chromosome 4 the malignant parent cell contains. Eleven
independent hybrid clones between SEWA and Rb(i6. i7)yBnr fibroblasts were
isolated, and, although all of them retained the one identifiable chromosome 4
derived from the SEWA cell, only one clone retained both the chromosomes 4
derived from the diploid fibroblast. Other clones had lost either one or both of the
fibroblast chromosomes 4. In this case, therefore, the diploid chromosomes 4 are
again selectively eliminated in vitro. The one clone (F) that had retained both the
fibroblast chromosomes 4 initially produced one tumour in nine mice with inocula
of 5 x io 5 cells per animal; but, on continued cultivation in vitro, the clone soon
became tumorigenic (Table 2). A re-examination of the karyotype after three weeks'
5-2
124 E. P. Evans, M. D. Burtenshaw, B. B. Brown, R. Hennion and H. Harris
cultivation in vitro showed elimination of one of the fibroblast chromosomes 4 in the
majority of the cells (Table 9). The tumours produced by this clone showed elimination of at least one fibroblast chromosome 4 in all cells. Another clone (D) showed,
on first examination, a duplication of the one identifiable chromosome 4 derived
from the SEWA cell and a total elimination of both chromosomes 4 derived from
the fibroblast. This clone, tested at the time of the karyological examination, gave
a 100% take incidence with inocula of 5 x io 5 cells per animal (Table 2). The
tumours produced showed no consistent further change in karyotype. While the
cytogenetic analysis of the SEWA hybrids is less satisfactory than that of the other
combinations we have described, they do provide a convincing explanation of the
instability of the suppression of malignancy in this cross.
Table 9. Chromosome 4 constitution of SEWA x Rb{ 16.17) jBnr
clone F and tumours derived from it
Copies of chromosome 4
per cell
SEWA
Expected in complete bi-parental hybrid
Clone F when first examined
(mean of 12 cells)
Clone F after 3 weeks' cultivation
(mean of 20 cells)
(Tumour 1 (mean of 10 cells)
Clone F - Tumour 2 (mean of 10 cells)
[Tumour 3 (mean of 10 cells)
RB7Bnr
NF
i»
2
1
1-9
85
74-S
1
1-2
69-1
1
I
1
09
1
I
70-2
70-0
69-1
#
Partial tandem translocation representing approximately 2 copies of chromosome 4 (see
text).
Hybrids in which suppression of malignancy cannot be demonstrated
TAjHaB xCBA/H-T9T6 fibroblast hybrids. Although crosses between TA3Ha,
the parental line from which TA3HaB was derived, and A.CA diploid fibroblasts
have in the past yielded occasional hybrid clones with reduced tumorigenicity
(Wiener, Klein & Harris, 1971), the majority of crosses between TA3HaB and
diploid fibroblasts are as tumorigenic as the TA3HaB parent cell. We have examined
five independent clones from a cross between TA3HaB and CBA/H-T 6 T 6 fibroblasts.
The cytogenetic analysis of these clones is shown in Table 10. Each of the five
clones contains two TA3HaB chromosome sets and one fibroblast chromosome set.
Since the probability that all these independently derived clones arose from the
chance fusion of two TA3HaB cells with one fibroblast is negligible, we consider it
likely that the extra TA3HaB chromosome set in these hybrids arose by duplication
of this chromosome set within an originally binucleate cell. Whatever the mechanism,
all of these hybrid clones initially have an excess of the chromosomes 4 derived
from the malignant parent cell, although there may be some reduction in the number
Analysis of malignancy. IX
125
of these chromosomes on continued cultivation in vitro. In three of the clones, both
chromosomes 4 derived from the fibroblast parent cell were eliminated; in the other
two clones, one of the fibroblast chromosomes 4 was eliminated. The tumorigenicity
of three of the hybrid clones was tested. All three gave 100% take incidences with
inocula of 5 x 10* cells per animal, and the latent periods before the appearance of
the tumours were comparable to that given by the parental TA3HaB tumour cell
(Table 2). The tumours produced by these hybrids contained no chromosomes 4
derived from the fibroblast parent cell. The failure to demonstrate suppression of
malignancy in this cross is thus no evidence for genetic dominance of malignancy.
In the TA3HaB x diploid fibroblast hybrids one finds not only rapid selective
elimination of the chromosomes 4 derived from the diploid parent cell, but also
a duplication of the chromosomes 4 derived from the malignant parent cell.
Table 10. Chromosome 4 constitution of five Ta^HaB x CBA/H-TBT6
hybrid clones
Copies of chromosome 4
per cell
Ta 3 HaB
Expected in complete 2Ta3HaB x CBA/
H-T.T, hybrid
Clone Ci (mean of 6 cells)
Clone A2 (mean of 8 cells)
Clone F2 (mean of 6 cells)
Clone D2 (mean of 11 cells)
Clone B2 (mean of 8 cells)
CBA/H-T.T,
NF
4
2
122
4
386
3
5'5
0
0
102-7
117-7
083
o-8
100-5
109-8
2
0
IO6-I
An exceptional clone
Of the many hybrid clones examined in this study, only one generated tumours
that failed to show selective pressure against the chromosomes 4 derived from the
normal fibroblast. One subpopulation of the YACIR IMP-x CBA/H-T 6 T 6 fibroblast clone 1G1 showed this erratic behaviour. As described above, clone 1G1
contained two copies of the chromosome 4 derived from the YACIR IMP~ parent
cell and one from the diploid fibroblast. It was highly tumorigenic and the tumours
initially produced by it did not differ in their chromosome constitution from the
cells injected. One subpopulation, tested after a period of growth in vitro, generated
tumours in which about half of the cells contained an additional chromosome 4
derived from the fibroblast parent cell. When one such tumour was serially transplanted, cells containing two fibroblast chromosomes 4 were still present in the
tumours produced in the 2nd and 3rd mouse passages. In this cell population the
diploid chromosome 4 thus behaves as if it had been derived from a malignant cell.
One explanation of this result might be that the single diploid chromosome 4
originally present in clone 1G1 had undergone, in this particular subpopulation,
some change that allowed it to escape the selective pressure directed against the
126 E. P. Evans, M. D. Burtenshatv, B. B. Brown, R. Hennion and H. Harris
diploid chromosomes 4 in all the other hybrids we have examined, including the
other YACIRIMP-xCBA/H-T 6 T 8 fibroblast hybrids. Such a change could be
produced either by a mutational event or by homologous crossing-over between the
fibroblast chromosome 4 and one of the malignant chromosomes 4.
DISCUSSION
The most important conclusion to be drawn from the present series of experiments
is that, for all the malignant tumours examined (carcinoma, melanoma, sarcoma and
lymphoma), suppression of malignancy involves the activity of some gene located
on the diploid mouse chromosome 4. This means that in all these tumours, the
chromosomes 4 in the malignant cell are in some way functionally different from
the chromosome 4 of the diploid cell. The cytogenetic evidence thus supports the
inference previously drawn from complementation tests, that malignant cells, even
though they are derived from different kinds of tumour, have some genetic lesion
in common (Wiener, Klein & Harris, 1974ft). The present study also provides
evidence for the view that the gene on the diploid chromosome 4 responsible for
the suppression of malignancy acts in a dose-dependent manner. In some crosses, the
loss of a single diploid chromosome 4 is enough to induce the re-appearance of
malignancy in cells in which it was initially suppressed; and this suppression can
also be overcome by an increase in the number of malignant chromosomes 4. In the
suppression of malignancy by cell fusion we are therefore titrating diploid chromosomes 4 against malignant chromosomes 4. A similar situation has been described
by Spira et al. (1981) for chromosome 15 in hybrids between diploid mouse fibroblasts and lymphoma cells from a T cell leukaemia of the AKR mouse. Jonasson
et al. (1977) had shown in previous work that, in certain crosses between malignant
and diploid cells, an increased dosage for some gene or genes located on chromosome
15 was required for proliferation of the hybrid cells in vitro; but we have not detected
any systematic changes in chromosome 15 in the present series of hybrids, which
includes crosses between the YACIR IMP- lymphoma and diploid fibroblasts. We
therefore think it probable that the involvement of chromosome 15 as described
by Spira et al. is limited to certain specific classes of lymphoid tumours; and it is
not, of course, excluded that the suppression of malignancy in these cell types
might also involve chromosome 4.
It is important to draw a distinction between the behaviour of chromosome 4 as
revealed by our studies and ' chromosome balance' models for the control of malignancy as proposed, for example, by Hitotsumachi, Rabinowitz & Sachs (1971). We
interpret our findings in terms of a classical gene-dosage effect for one particular
gene on one particular chromosome. Hitotsumachi et al. propose that malignancy is
determined by a balance between the co-operative activity of one group of chromosomes and the co-operative activity of another group of different chromosomes. We
see nothing in our data to support a ' chromosome balance' model of this type.
Since the suppression of malignancy in hybrid cells can be overcome either by
the loss of diploid chromosomes 4 or by an increase in the number of malignant
Analysis of malignancy. IX
127
chromosomes 4, the functional abnormality specified by the malignant chromosome
4 cannot be the absence of some normal gene product, such as might result, for
example, from the deletion of a structural gene. In the latter case, no increase in
the number of malignant chromosomes 4 could have any effect. The present observations also have some bearing on models of malignancy that require the overproduction of some normal gene product. If malignancy were due to the overproduction of a normal gene product, then the suppression of malignancy in hybrid
cells must involve the abolition, or at least the substantial reduction, of this overproduction: the diploid genome in the hybrid cell must exercise some form of
regulatory control of which the malignant genome is no longer capable. The overproduction of the normal gene product in the malignant cell thus implies some
functional impairment elsewhere in the genome. We would therefore argue that,
whether malignancy is due to the overproduction of a normal gene product or to
the production of an abnormal gene product, its suppression in hybrid cells involves
the restoration of some genetic function that is defective in the malignant cell.
The experiments we have described seem to us to resolve without difficulty most
of the conflicting observations that have been made concerning the suppression of
malignancy in hybrid cells. Aviles, Jami, Rousset & Ritz (1977) and Aviles, Ritz &
Jami (1980) claim that hybrids between malignant and diploid mouse cells, produced
either in vivo or in vitro, generate tumours that may contain at least one copy of
each of the chromosomes derived from the diploid parent cell. These authors therefore conclude that no single diploid chromosome can be responsible for the suppression of malignancy. However, the present experiments show that the elimination
of only one of the pair of diploid chromosomes 4 may result in the reappearance of
malignancy in hybrid cells in which it was initially suppressed. Indeed, the results
obtained by Aviles et al. (1980) closely resemble those reported here for the A9HT
crosses. The malignant parent cell used by Aviles et al. was clone iD, which, like
A9HT, is a derivative of the L cell line. As reported, it contains a metacentric
chromosome composed of two copies of chromosome 4, but, in addition to this
metacentric chromosome, it also contains an acrocentric chromosome 4, which, in
the absence of an appropriate morphological marker, cannot be distinguished from
a normal chromosome 4. Tumours produced from crosses between clone iD and
diploid cells would thus contain an excess of malignant chromosomes 4, even if
they had retained both copies of the diploid chromosome 4. Aviles et al. observed
that the great majority of the cells in the tumours produced by their hybrids had
eliminated one copy of the diploid chromosome 4. We would argue that the loss of
diploid chromosomes 4 might have been even greater, since at least one of the
acrocentric chromosomes 4 classified by Aviles et al. as diploid might have originated
from the malignant clone iD parent cell. Kucherlapati & Shin (1979) also found
that, in crosses between malignant mouse cells (one of which was again clone iD)
and diploid human fibroblasts, each chromosome was represented in at least one
copy in the range of tumours produced by the inoculation of the hybrid cells; and
these authors drew the same conclusion as Aviles et al. As the studies of Klinger
et al. (1978) show, the analysis of malignancy in crosses between malignant rodent
128 E. P. Evans, M. D. Burtenshaw, B. B. Brozon, R. Hennion and H. Harris
cells and diploid human cells is difficult, due to the preferential, but variable, elimination of the human chromosomes. However, in the light of our present findings,
there is no reason to exclude the possibility that in such interspecific hybrids the
loss of a single copy of one particular human chromosome might also be enough to
induce the reappearance of malignancy. The recent study of Stanbridge, Flandermeyer, Daniels & Nelson-Rees (1981) suggests that this may indeed be the case for
human chromosone 11 or human chromosome 14. The experiments of Schafer et al.
(19S1), in which no suppression of malignancy was observed in crosses between
malignant Chinese hamster cells and diploid mouse cells, resemble our experiments
with the TA3HaB carcinoma. All the tumours produced by the hybrids studied by
Schafer et al. contained the genome of the malignant parent cell in at least two copies.
There remains one set of observations that cannot be accommodated in our present
proposal, namely, the claim by Croce, Aden & Koprowski (1975 a, b) and Koprowski
& Croce (1977) that a single human chromosome carrying the simian virus 40
genome renders normal mouse macrophages malignant in a genetically dominant
manner. Since other workers have found that malignancy can readily be suppressed
when cells transformed by simian virus 40, and other oncogenic viruses, are fused
with diploid cells (Gee & Harris, 1979; Howell & Sager, 1979; Pereira-Smith &
Smith, 1981; Marshall & Dave, 1978), and since serious technical criticisms have
been made of the Croce and Koprowski experiments (Gee & Harris, 1979; Harris,
1979), it seems reasonable to propose that their observations be confirmed before
they are accepted as a decisive counter-argument. The analysis of crosses between
myelomas and diploid cells remains to be done. The experiments of Giacomoni
(1979) are uninformative in that they do not contain the appropriate karyological
information. Some preliminary work that we have done with myeloma hybrids leads
us to suppose that this analysis will not be easy, due to the unpromising nature of
the karyotype of most of the available myelomas.
E.P.E. and M.D.B. are members of the external staff of the Medical Research Council.
B.B.B. received a giant from the E.P.A. Research Fund. The work was generously supported
by the Cancer Research Campaign. We thank Mr J. H. D. Kent for skilful technical assistance.
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{Received 1 March 1982)