y. Cell Sci. 16, 189-198 (1974)
Printed in Great Britain
189
THE ANALYSIS OF MALIGNANCY
BY CELL FUSION
VI. HYBRIDS BETWEEN DIFFERENT TUMOUR CELLS
F. WIENER, G. KLEIN
Department of Tumour Biology, Karolivska
104 01 Stockliolm 6o, Sweden
Institutet,
AND H. HARRIS
Sir William Dunn School of Pathology, University of Oxford,
Oxford OXi jRE, England
SUMMARY
Previous studies with a variety of transplantable mouse tumours showed that in hybrids
between malignant and non-maJignant cells, malignancy behaved as a recessive character:
the hybrid cells, so long as they retained something close to the complete parental chromosome
sets, had little or no ability to grow progressively in vivo. In the experiments we now describe
the heritable lesions determining the malignant phenotype were further explored by complementation analysis in which the various tumour cells were fused with each other. Forty-two
clonal populations derived from twelve crosses between different kinds of rumour cells were
examined. Only one cross generated hybrid cells with reduced tumorigenicity: in all other
cases the hybrid cells formed were highly malignant. It thus appears that, in a wide range of
different tumours, the lesions determining the malignant phenotype, although recessive, fail to
complement each other.
INTRODUCTION
We have explored in a wide range of mouse material the consequences of fusing
a malignant cell with a non-malignant or less malignant one (Harris et at. 1969; Klein,
Bregula, Wiener & Harris, 1971; Bregula, Klein & Harris, 1971; Wiener, Klein &
Harris, 1971; Harris, 1971; Klein, Friberg, Wiener & Harris, 1973; Wiener, Klein &
Harris, 1974). The results obtained are in principle very simple. Whenever a malignant
cell is fused with a cell of low tumorigenicity or with a normal diploid cell, isolated
directly from the animal, the hybrid cells show a dramatic reduction in tumorigenicity
compared with the malignant parent cell. Further, we have presented strong evidence
for the view that those tumours that are produced by the injection of hybrids between
malignant and non-malignant cells arise not by the growth of the hybrid cell population as a whole, but by the selective overgrowth of cells from which certain specific
chromosomes have been eliminated. The cells that give rise to tumours in crosses of
this kind are thus, in general terms, segregants of the original hybrid cell population.
If, then, the lesion or lesions determining the malignant phenotype are genetic
ones, they may be described operationally as recessive to the normal non-malignant
wild type. Claims to the contrary have not been substantiated. Earlier experiments
190
F. Wiener, G. Klein and H. Harris
purporting to show that malignancy was a dominant trait in hybrids between malignant and non-malignant cells foundered on an inadequate appreciation of the role of cell
selection in the formation of a tumour (Barski, Sorieul & Cornefert, 1961; Scaletta &
Ephrussi, 1965; Defendi, Ephrussi, Koprowski & Yoshida, 1967); and the authors
of these experiments now appear no longer to adhere to their original interpretation
(Barski, 1970; Ephrussi, 1972). A later report in which hybrids between malignant
and non-malignant hamster cells were said to produce tumours composed of cells that
had not undergone chromosome losses (Berebbi & Barski, 1971) has since been shown
to be erroneous (Berebbi, Lepoint, Fondarai & Meyer, 1973). The literature does not
at present contain a single convincing example of a cross between a malignant and a
non-malignant cell in which the malignant phenotype has been shown to be dominant.
This being so, it seemed of interest to attempt a complementation analysis of the
lesions determining the malignant phenotype, since this form of analysis has been
found informative in the study of other heritable recessive lesions in somatic cells
(Kao & Puck, 1972). In the present paper we report the results of such an analysis.
The tumour cells that had previously been fused with non-malignant cells were fused
with each other, and the tumorigenicity of the resulting hybrid cells was measured.
(Some of these hybrids have been described in other contexts in previous papers; they
are included here in order to permit a complete survey to be made of all crosses that
we have made between one malignant cell and another.)
MATERIALS AND METHODS
Cells
The following tumour cells were studied: SEWA, a polyoma virus-induced sarcoma;
MSWBS, a methylcholanthrene-induced sarcoma; MBA, another methylcholanthrene-induced
sarcoma; A9HT, a highly malignant sarcoma selected from the Ao. line (an L cell derivative) by
passage through the animal; YAC, a Moloney virus-induced lymphoma; EL4, another Moloney
virus-induced lymphona; YACIR, a derivative of YAC selected for reduced expression of the
Moloney virus-induced cell surface antigen by passage through mice immunized against the
Moloney virus; TA3 Hauschka (TA3Ha), a spontaneous mammary carcinoma with reduced
expression of H-2 isoantigens; TA3Ha B, a clonal subline of TA3Ha selected for resistance to
6-thioguanine and lacking inosinic acid pyrophosphorylase activity; the Ehrlich ascites tumour.
Precise details of the origins and properties of these tumours have been given previously (Klein
et al. 1971; Harris, 1971; Klein et al. 1973; Wiener, Klein & Harris, 1973).
Cell fusion and selection of hybrids
The cells were fused together by means of inactivated Sendai virus essentially as described by
Harris & Watkins (1965). The lymphoma cells, which fuse less readily than the carcinoma and
sarcoma cells, were added to the mixed cell suspension at a concentration 5-10 times greater
than that of the other cell in the combination. Various selective procedures were used to obtain
the hybrid cell populations. Where one of the parent cells lacked inosinic acid pyrophosphorylase
(TA3Ha B and A9HT), the hybrid cells were selected in HAT medium which eliminates cells
lacking this enzyme. Cells that do not attach, or attach poorly, to glass or plastic surfaces
(YAC, YACIR, EL4, SEWA) were eliminated by frequent medium changes. MSWBS grows
poorly under conventional cultural conditions, so that its removal does not present a problem.
Where the two parent cells did not embody readily selectable markers, differences in surface
properties between the parent cells and the hybrid were exploited to select for the latter. All
these selective procedures have been described in detail in the papers listed above.
Analysis of malignancy by cell fusion. VI
191
Cytological and immunological tests
Chromosome preparations of air-dried metaphase plates were made in the usual way (Rothfels
& Siminovitch, 1958; Klein et al. 1971). H-2 isoantigens on the surface of the hybrid cells were
measured by quantitative immune adsorption tests as previously described (Klein, Friberg &
Harris, 1972).
Assay for tumorigenicity
All tests were done, as before, in genetically compatible newborn mice given 4 J kg"1 of total
body X-irradiation. Inocula of approximately 2 x io5 to 2 x 10* cells were injected subcutaneously. Animals were scored as negative if they failed to produce a tumour within
3 months.
RESULTS
Identification of hybrid cells
The hybrid cells were identified by their chromosomal constitution and/or by their
immunological properties. Ehrlich, A9HT, MSWBS, MBA and SEWA had readily
identifiable chromosome markers, so that the hybridity of crosses between these cells
could be confirmed decisively by total chromosome number and by the presence of
Table 1. Chromosomal constitution and histocompatibility isoantigens of
parent tumour cells
Cell type
SEWA
MSWBS
MBA
A9HT
YAC
EL4
YACIR
TA3Ha
TA3Ha B
Ehrlich
H-2
Modal
chromosome
complex
no.
Range
H-2*
H-2*
H-2 k
H-2 k
H-2°
H-2 b
H-2'
H-2*
H-2'
43
29
—
42-44
28-30
80-106
S3
47-57
40
39-41
38-40
40-42
40-42
40-42
71-94
?
39
40
40
40
76
Marker
chromosomes
Yes
Yes
Yes
Yes
No
No
No
No
No
Yes
markers from both parent cells. Where the parent cell did not possess a chromosome
marker, identification of the hybrid was made from the total chromosome number and
the presence of the appropriate parental H-2 isoantigens. All hybrid cells tested were
positively identified by one or both of these procedures. Table 1 shows the chromosomal constitutions and immunological properties of the parental cells, and Table 2
the chromosomal constitutions and immunological properties of the hybrids derived
from them.
F. Wiener, G. Klein and H. Harris
192
Table 2. Chromosomal constitution and histocompatibility isoantigens of
hybrid cells
Marker chromosomes
from
Cell type
chromosome no. Range
SEWA/A9HT
Clone 1
5
SEWA/TA3Ha B
Clone 1
3
4
MSWBS/A9HT
Clone 1
2
3
4
5
MSWBS/TA3Ha
Clone 2
3
4
5
6
MSWBS/EL4
Clone 1
72
80
83
81
79-84
78-85
74-85
79
77
74
75-82
71-86
64-84
73-80
73-80
ASW
75
78
70
66
6
MBA/YACIR
Clone 1
2
67
2
3
5
Ehrlich/AgHT
Clone 1
3
4
6
C57BI
69-73
69-73
68-74
66-72
ASW
67
66-72
64-69
68
67
69
67
39-79
45-76
56-93
38-70
71
ASW
CBA
125
122
93-135
65-161
YACIR/A9HT
Clone 1
C3H
63-74
62-74
64-71
64-74
62-75
ASW
70
71
4
5
C3H
ASW
69
67
69
70
5
Parent 2
ASW
3
5
MSWBS/YACIR
Clone 3
Parent 1* Parent 2 Parent I*
ASW
61-82
72-81
74; 77; 79
2
MSWBS/YAC
Clone 2
H-2 isoantigens
from
C3H
78
73
104
93
75-90
72-82
92-107
80-97
C3H
no
105
92-125
97-119
92
92-111
102
102-117
Analysis of malignancy by cell fusion. VI
193
Table 2 (cont.)
Cell type
TA3HB/A9H.T
Clone 4
5
6
TA 3 HaB/EL 4
Clone if
2
3
Modal
chromosome no. Range
Marker chromosomes
H-2 isoanrigens
from
from
A
,
,
,
*
,
Parent i* Parent 2 Parent i* Parent 2
91
94
91
87-95
90-95
89-95
•
.
.
+
+
+
120
77
66
105-131
71-81
61-70
.
.
.
.
.
.
A
+
+
+
A
+
+
+
C3H
•
.
.
C57BI
—
+
.
• First parental cell mentioned in the combination, e.g. SEWA in SEWA/A9HT.
f Triparental hybrid.
Growth of hybrid cells in vivo
For all combinations clonal populations were tested. For SEWA/A9HT, SEWA/
TA 3 HaB, EL 4 /TA 3 HaB, MSWBS/A9HT, YACIR/A9HT and Ehrlich/AgHT,
primary clones derived from independent fusion events were studied. For MSWBS/
TA3HaB, MSWBS/EL4, MSWBS/YAC, MSWBS/YACIR and TA 3 Ha/A 9 HT,
primary clones could not be isolated and the tests were done on secondary clones
derived from early wild type populations. The results of the take incidence tests in
genetically compatible X-irradiated newborn mice are given in Table 3. It will be seen
that in all combinations except MSWBS/YACIR the take incidences were not far
short of 100%. For MSWBS/YACIR, a marked reduction in take incidence relative
to the 2 parental cells was observed in all clones.
Tumours produced from the hybrid cells
We have previously shown that the chromosomal constitutions of tumours produced
from hybrids between the malignant A9HT line and other malignant cells do not
differ significantly from those of the hybrid cells injected (Wiener et al. 1973). This is
in marked contrast to the findings obtained with hybrids between malignant and nonmalignant cells, where tumours are always the result of strong selection for segregant
cells which have eliminated certain chromosomes. Tumours produced from some of
the other malignant x malignant crosses described in the present paper were also
examined. The results again showed that the chromosome constitutions of the
tumours did not differ significantly from those of the hybrid cells injected. Table 4
summarizes the results obtained with MSWBS/TA3Ha and MSWBS/EL4 hybrids.
MSWBS/YACIR, being the only case in which the fusion of 2 malignant cells gave
rise to hybrids of reduced tumorigenicity, was subjected to intensive study. The
detailed analysis of these hybrids and the tumours derived from them will be the
subject of later communications. In the present context we simply report that the
II
C E L 16
F. Wiener, G. Klein and H. Harris
194
Table 3. Growth of tumour x tumour hybrids in vivo
Cell type
SEWA/A9HT
Clone 1
3
5
Total
SEWA/TA3Ha B
Clone 1
3
4
Total
MSWBS/A9HT
Clone 1
2
3
4
5
Total
MSWBS/TA3Ha
Clone 2
3
4
S
6
Total
MSWBS/EL4
Clone 1
3
5
8
Total
Genotype of host
A.SWxCBA
or
A.SWxC,H
F1 hybrids
A.xA.SW
F1 hybrids
Percentage
takes
19/21
14/14
14/14
47/49
96
IS/15
II/II
14/14
40/40
A.SWxCBA
or
A.SWxC,H
Fx hybrids
A.xA.SW
F1 hybrids
100
3/3
10/10
54/55
4/4
7/7
78/79
99
25/25
61/61
9/9
29/31
26/26
150/152
A.SWXC57BI
.?! hybrids
99
40/40
7/7
20/20
16/16
MSWBS/YAC
Clone 2
5
Total
A.xA.SW
•Fj hybrids
MSWBS/YACIR
Clone 3
4
5
6
A.xA.SW
-f7! hybrids
Total
No. takes
(total no. animals
with progressive
tumours/total
no. inoculated)
83/83
100
23/23
37/37
60/60
100
60/89
39/8i
88/120
63/104
25O/394
Analysis of malignancy by cell fusion. VI
195
Table 3 (cont.)
Cell type
MBA/YACIR
Clone 1
2
Genotype of host
CBAxA.
•F] hybrids
Total
YACIR/AoHT
Clone i
2
3
5
Total
Ehrlich/AgHT
Clone 1
3
4
A. x CBA
or
A.xCH
Ft hybrids
A. x CBA
or
A.xC,H
F, hybrids
Total
TA3Ha B/EL4
Clone 1
2
3
Total
21/21
94
46/50
55/55
38/38
I39/H3
A.XC57BI
JF1! hybrids
98
37/41
74/77
34/39
166/178
Total
96
31/31
13/13
22/25
71/71
137/140
C3H and C,H
Ft hybrids
Percentage
takes
179/182
50/56
229/238
6
TA3Ha/AoHT
Clone 4
5
6
No. takes
(total no. animals
with progressive
tumours/total
no. inoculated)
97
60/60
50/50
52/52
162/162
100
tumours derived from these hybrids showed marked variation in chromosome number
and included many cases in which the modal chromosome number was substantially
reduced.
DISCUSSION
Except in the case of MSWBS/YACIR, there was no significant reduction in
tumorigenicity in any of the hybrids produced by fusing the different tumour cells
with each other. A cross between one malignant cell and another is thus, in general,
malignant. If, then, we are dealing with recessive genetic: lesions in the determination
of the malignant phenotype, these lesions fail to complement. MSWBS/YACIR is,
at a first glance, an exception, but it is clearly a very special case. YACIR was derived
13-2
196
F. Wiener, G. Klein and H. Harris
Table 4. Chromosomal constitutions of hybrids and tumours derived from them
No. 1Diarmed
chromosomes
Total
chromosome no.
Cell type
Genotype of
test animal
A
Mode
Range
69
66
r
Mode
Range
63-74
10
61-71
10
67
66
67
62-74
10
8-12
9-12
9-11
9-11
9-11
70
MSWBS/TAaHa
Clone 2
In vitro
A. xA.SW
3
In vitro
5
A. xA.SW
A. xA.SW
In vitro
A. xA.SW
In vitro
A.xA.SW
6
69
66
68
65-69
10
63-70
10
64-74
66-74
11
62-75
64-70
10
69-73
67-72
67-74
66-72
67-72
12
11
10
9-13
9-12
9-13
9-11
MSWBS/EL4
Clone 1
In vitro
A.SWXC57BI
A.SWXC57BI
5
In vitro
A.SWXC57BI
70
72
69
67
69
12
12
11
11
11-13
10-13
10-13
9-12
9-12
by immunological selection from YAC, and must therefore initially have had the same
genetic lesions as YAC; but MSWBS/YAC hybrids show no reduction in tumorigenicity. Moreover, both MSWBS and YACIR fail to complement with A9HT and
should therefore, on this model, bear lesions at the same locus. We do not yet have a
satisfactory explanation for the reduced tumorigenicity of MSWBS/YACIR; but it is
for the moment difficult to accept this reduced tumorigenicity as strong evidence for
a second complementation group. If, then, non-complementation in differentiated
somatic cells has the same biological significance as non-complementation in other,
simpler, organisms, we are faced with the conclusion that malignancy, as denned by
our test system, is determined in a wide range of different tumours by a recessive
lesion at a single locus.
This conclusion is so startling that it must be, and obviously will be, treated with
great reserve. Other explanations are possible. If the malignant phenotype were
determined not by mutational events, but by 'epigenetic' derangements, then the
complementation analysis would be extremely difficult to interpret, and it would not
necessarily provide any information about the identity or non-identity of the loci
involved. Although it is clear from the presence of specific marker chromosomes that
the great majority of the tumours with which we are dealing are clonal in origin, and
from the cell fusion studies that the expression or non-expression of the malignant
phenotype is chromosomally determined, no experimental evidence yet permits one
to decide between 'genetic' and 'epigenetic' models for the generation of malignancy.
And even if malignancy were determined by genetic events of a mutational type, it is
conceivable that in hybrid somatic cells genes capable of complementing each other
Analysis of malignancy by cell fusion. VI
197
might fail to do so because they are maintained in an inactive state by stable 'epigenetic' mechanisms akin to those that determine heritable differentiation or the
inactivation of the X chromosome. If this were the case, failure to complement would
not necessarily imply that the genetic lesions were at the same locus. It is not difficult
to construct 'epigenetic' models, or models combining 'genetic' and 'epigenetic'
elements, which would adequately explain our findings. The range of possibilities
would, however, be drastically reduced if the lesion determining the malignant
phenotype could be assigned by cytogenetic or linkage analysis to a specific chromosome, and reduced even further if the same chromosome were found to be involved
in a range of different tumours. We have already explained how the segregation of
chromosomes in tumours produced from malignant x non-malignant crosses might be
used to facilitate such an analysis (Wiener et al. 1974); and we are exploring this and
other possibilities in a range of different hybrids. Until this analysis is complete, we
limit our claim to the statement that if malignancy is determined by genetic events of
a mutational kind these behave as recessives in hybrid cells, and they do not, in a wide
range of different crosses, complement each other.
The work at Stockholm was supported by grants from the Swedish Cancer Society, the
National Cancer Institute of the U.S. Public Health Service and the Damon Runyon Memorial
Fund; the work at Oxford by the Cancer Research Campaign.
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