Download A gene for the suppression of anchorage independence is located in

A gene for the suppression of anchorage independence is located in rat
chromosome 5 bands q22-23, and the rat alpha-interferon locus maps at the
same region
M. QUAMRUL ISLAM 1 , JOSIANE SZPIRER 2 , CLAUDE SZPIRER 2 , KHALEDA ISLAM 1 ,
JEAN-FRANCOIS DASNOY 2 and GORAN LEVAN 1 '*
' Department of Genetics, University of Gothenburg, Box 33031, S-400 33 Gothenburg, Sweden
Department of Molecular Biology, Universite Libre de Bmxelles, Belgium
2
•Author for correspondence
Summary
Cell hybrids between malignant mouse hepatoma
cells and normal rat fibroblasts -with approximately
one set of chromosomes from each parent
exhibited remarkable karyotypic stability. Most
chromosomes of both parents were retained even
after prolonged culture in vitro. Normally, such
hybrids showed suppression of the transformed
phenotype and formed no colonies in soft agar.
However, two hybrids, BS140 and BS181, formed a
few colonies in soft agar when many cells were
seeded, and also occasional foci of cells were
detected piling up in monolayer cell cultures. We
isolated soft agar colonies (a-subclones) and subclones from foci (h-subclones) of both hybrids, and,
as a control, subclones of cells from random areas
without foci of one hybrid (BS181 p-subclones).
When tested for soft agar growth, cells from the aand h-subclones of both BS140 and BS181 formed
colonies at frequencies comparable to the malignant mouse hepatoma parent, whereas the control
cells of the BS181 p-subclones (like the normal rat
parental cells) yielded no soft agar colonies. All the
cell lines -were subjected to detailed karyotype
analysis in G-banding, which resulted in the
finding that cells from the original BS140 hybrid
contained at least one copy of each rat chromosome, whereas BS140 a- and h-subclones had lost
both copies of rat chromosome 5. Similarly, the
original BS181 hybrid contained at least one copy of
each rat chromosome, whereas BS181 a- and
h-subclones displayed a deletion of the segment
q22-23 of rat chromosome 5. In contrast, the control
BS181 p-subclones contained one or two copies of
non-deleted rat chromosome 5. The conclusion is
that a gene for the suppression of anchorage independence is located in the segment 5q22-23. We
propose to call this gene SAI1 (for suppression of
anchorage independence).
Using Southern blotting, we tested whether any of
several gene probes, known to correspond to DNA
sequences in rat chromosome 5, were homologous
to sequences in the deletion. Only one probe, corresponding to the active alphai-interferon gene, was
shown to be located within the deletion. Hence, the
SAI1 gene is closely linked to the alphai-interferon
gene, and might be identical to this locus.
Introduction
hereditary cancers such as retinoblastoma (Knudson,
1985), where both normal alleles have to be inactivated
for the malignant phenotype to be expressed (Cavenee et
al. 1983). The occurrence of emerogenes has also been
inferred from another line of research, i.e. the study of
hybrids between malignant and normal somatic cells.
Many workers have shown that in such hybrids the
tumorigenic phenotype is suppressed, as determined by
lack of ability to form tumours in syngeneic animals or in
nude mice (Harris et al. 1969; Wiener et al. 1971, 1974;
Stanbridge, 1976; Marshall & Dave, 1978; Sager &
A group of genes with the ability to suppress the
malignant phenotype in mammalian cells has been identified. These genes have been called anti-oncogenes or
tumour-suppressor genes, and the inactivation of both
alleles of such genes seems to be a requirement for
malignant transformation in many instances. Following
the suggestion of Todaro (1988), we will call these genes
emerogenes. The dominant action of normal alleles of
emerogenes is convincingly illustrated by the study of
Journal of Cell Science 92, 147-162 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Key words: tumour suppression, emerogene, anchorage
independence, alpha-interferon.
147
Kovac, 1978; Stanbridge et al. 1981, 1982; Benedict et
al. 1984). It has usually been possible, however, to
isolate, from the suppressed hybrid population, single
cells that have regained the tumorigenic phenotype. Such
revertants display lower chromosome counts than the
original hybrid and it has been concluded that the
reversion is due to loss of chromosomes that carry tumour
suppressor genes (emerogenes); these studies led to the
conclusion that such genes are located on mouse chromosome 4 (Jonasson et al. 1977; Evans et al. 1982), on
Chinese hamster chromosome 3 (Craig et al. 1988) and
on human chromosomes 1 and 11 (Benedict et al. 1984;
Kaelbling & Klinger, 1986; Srivatsan et al. 1986; Saxon
et al. 1986). This field has recently been reviewed by
several authors (Sager, 1985, 1986; Stanbridge, 1985;
Harris, 1985, 1986; Klein, 1987).
In vitro studies have shown that, at least in some
hybrids, transformation-associated
properties like
anchorage independence are also suppressed in hybrids
formed between transformed, malignant cells and normal
cells (Marshall & Dave, 1978; Marshall, 1980; Szpirer &
Szpirer, 1980; Marshall & Sager, 1981; Dyson et al.
1985; Stoler & Bouck, 1985). However, tumorigenicity
and transformation can be dissociated, since some
hybrids formed between tumour and normal cells remain
transformed but are non-tumorigenic (see e.g. Straus et
al. 1976; Stanbridge & Wilkinson, 1978).
Obviously, it is of great interest to identify the chromosomes that carry tumour- or transformation-suppressor
genes. Most investigations in this field have been carried
out with intraspecific hybrids (Wiener et al. 1971; Sager
& Kovac, 1978; Klinger, 1980; Stanbridge et al. 1981).
In these studies a major problem has been to distinguish
unequivocally between the chromosomes of the abnormal
and of the normal parental cells. In order to circumvent
this problem various approaches have been attempted,
such as utilizing translocation chromosomes, variations of
C-bands among strains, isozyme variations, or restriction-fragment length polymorphisms (RFLPs). Of
necessity, these methods focus on but one or a few of the
chromosomes. From the viewpoint of refined chromosome identification, interspecific hybrids should be a
much more workable material, but in this case, there is
often the disadvantage of karyotype instability in the
hybrids and selective loss of chromosomes from one of
the parental species.
In the present investigation, we have studied interspecific hybrids between malignant, transformed mouse
cells and normal embryonic rat skin fibroblasts. These
hybrids proved to have remarkably stable chromosome
constitutions, and therefore provided a means of determining the derivation of all the chromosomes whether
from the malignant or the non-malignant parental cell.
Hybrids containing approximately one set of chromosomes from each of the parental cells were shown to be
suppressed for the transformed phenotype, i.e. to be
anchorage-dependent. From the primary, non-transformed hybrids, anchorage-independent sublines with
limited chromosome loss could be isolated. The chromosome analysis of these hybrids indicated clearly that a
suppressor of anchorage independence was located in a
148
M. Q. Islam et al.
specific region of rat chromosome 5. We also showed that
the rat alpha-interferon locus was located in the same
region.
Materials and methods
Parental and hybrid cells
The BWTG3 cell line is a clonal derivative from a hepatoma,
which arose spontaneously in a mouse of the C57BL/J strain.
The BWTG3 clone was isolated after selection in 6-thioguanine
and is deficient in the HGPRT enzyme. Rat skin fibroblasts
(RSF) were obtained from skin of embryos of the SpragueDawley strain and were cultured for about 2 weeks before
fusion.
The derivation of the BS series of mouse-rat hybrids has
been described (Szpirer & Szpirer, 1979). Briefly, mouse
BWTG3 and rat skin fibroblasts were harvested by trypsinization, rinsed in culture medium without serum, and fused in
suspension using u.v.-irradiated Sendai virus. Hybrids were
selected in HAT-medium (Littlefield, 1964). In this medium
BWTG3 cells are effectively killed. The normal rat fibroblasts
do not form any distinct colonies and the HAT-resistant hybrid
clones (one per dish) could be isolated by means of stainless
steel cylinders about 2 weeks after fusion.
Growth in soft agar
Cells were tested for growth in medium containing 0 - 3 % agar
(Noble agar, Difco) by the method of MacPherson & Montagnier (1964). Two or three replicate plates were inoculated
with 102—106 cells. The dishes were fed every week with 1 ml of
medium. Colonies larger than 0-1 mm across were counted after
4 weeks of incubation at 37°C in a humidified CO2 incubator.
As a control, the plating efficiency of cells tested for growth in
agar was determined by plating 10 —10 cells in 5 ml of medium
in 60 mm dishes: 1 ml of medium was added to each dish after 7
days, and the number of colonies was counted after about 2
weeks.
Chromosome analysis
Chromosome preparations were made and G-banded according
to the standard techniques of our laboratory (Martinsson et al.
1982; Islam & Levan, 1987). Complete karyotypes were
arranged from cutout chromosomes of enlarged photographic
prints. At least 10 cells were karyotyped from each line studied.
The mouse chromosome nomenclature recommended by the
Committee on Standardized Genetic Nomenclature for Mice
(1972) and by Nesbitt & Francke (1973) was followed. For rat
chromosomes, the chromosome nomenclature recommended
by the Committee for a Standardized Karyotype of Rattus
norvegicus (1973) and the G-banding nomenclature of Levan
(1974) were applied.
Southern blot analysis
This was done as described by Southern (1975). The probes
were labelled according to the method of Feinberg & Vogelstein
(1984), using the multiprime DNA labelling system (Amersham). The alpha-interferon probe was the pPCl plasmid,
containing a 2-Skb EcoRl fragment, which includes the rat
alphapinterferon gene (Dijkema et al. 1984).
Results
General characteristics of BS cell hybrids
Two types of hybrids emerged from the cell fusion
experiments between BWTG3 mouse hepatoma cells and
normal rat skin fibroblasts. Type I hybrids contained
about 100 chromosomes representing approximately the
sum of the chromosome numbers of the parental cells less
10% (Szpirer & Szpirer, 1979). Specifically, the average
number of rat chromosomes in these hybrids was between 32 and 38. As has been described (Szpirer &
Szpirer, 1980) the type I BS hybrids resembled the nontransformed rat fibroblast parents in that they grew to a
relatively low saturation density and they did not form
colonies in soft agar: they clearly exhibited suppression of
the transformed phenotype. Type II displayed higher
total chromosome numbers (averages between 122 and
193 in different type II hybrids) and also fewer rat
chromosomes (averages between 12 and 27). The relative
contribution of genetic material from the hepatoma
mouse parent was thus considerably greater in the type II
hybrids, and they proved to grow to higher saturation
densities on plastic and to have cloning efficiencies in soft
agar that resembled that of the BWTG3 cells; these
hybrids were not suppressed for the transformed phenotype (Szpirer & Szpirer, 1980).
Growth characteristics of two type I hybrids: BSJ40
and BS181
As stated above, most type I hybrids were unable to form
colonies in soft agar. Two hybrids, BS140 and BS181,
however, yielded a few soft agar colonies when many cells
were plated. Such rare colonies were isolated and propagated separately (BS140 a-series and BS181 a-series).
Furthermore, in these two hybrids foci of cells piling up
could be distinguished in confluent culture bottles. Such
foci of high cell density were isolated, subcloned, and
propagated separately (BS140 h-series and BS181
h-series). In addition, as a control, BS181 cells were
subcloned on plastic, and subclones picked at random
(BS181 p-series). Each of the resulting lines was tested
for growth in soft agar, and it was found that the a-series
as well as the h-series of subclones would form colonies in
soft agar at frequencies comparable to that of the malignant mouse parent, whereas cells from the p-series were
completely unable to form such colonies. The results
have been compiled in Table 1.
Chromosome analysis
General findings. In order to determine whether the
transformed a- and h-subclones derived from BS140 and
BS181 had lost specific chromosomes, the chromosome
constitution of these cells was determined in detail and
compared with that of the parental, non-transformed
hybrids and with that of the control, non-transformed
subclones of the BS181 p-series. At least 10 complete
karyotypes were analysed from each cell line studied.
Average total chromosome numbers as well as numbers of
mouse- and rat-derived chromosomes are listed in
Table 2. From this table it is evident that the loss of
chromosomes was quite limited, and that chromosomes
had been lost from both parental cell types to approximately the same degree. Tables 3 and 4 give data on the
average number of each mouse chromosome per cell:
normal mouse chromosomes in Table 3 and mouse-
Table 1. Cloning efficiency of the mouse and rat
parental cells and of the derived cell hybrids
Cloning efficiency
Cell line
Parents
BWTG3
RSF
Hybrids
BS140
On plastic (%)
In soft agar (%)
30
2xlO~ 3
9
<10~ 5
19
<5xl0~5
0-05
1-5
0-15
1
5
2
BS140hl
BS140h2
BS140h3
BSHOalO
BS140a20
BS140a31
20
13
25
15
30
25
BS181
BS181H1
14
BS181a2
BS181a3
BS181a4
BS181a5
BS181plO
BS181pll
35
30
25
10
22
40
10
2xlO~ 3
3
10
0-2
1
5
<2xlO-4
<2xlO-4
derived markers in Table 4. In Table 5 the average
numbers of rat chromosomes per cell have been recorded.
Since the occurrence of rat-derived marker chromosomes
in these lines was quite limited, we have not made a
separate table for them. In Table 5 these markers have
been included among the normal chromosomes from
which they were derived. The kinds of rat chromosomal
markers found were translocations, deletions and duplications. Most of them were seen in single cells only. A
detailed description of the rat markers is given in Table 5.
Loss of rat chromosome 5 material in the anchorageindependent sublines. The cells of the BS140 hybrid
clone contained at least one copy of each mouse and rat
chromosome (Fig. 1). The anchorage-independent subclones of BS140 (the h- and a-series) had very similar
karyotypes except for one significant deviation: they
contained no copy of rat chromosome 5. Sample karyotypes from BS140h3 and BS140a20 are given in Figs 2
and 3.
The cells of the BS181 hybrid clone also contained at
least one copy of every mouse and rat chromosome
(Fig. 4). All hybrids of the BS181 h-series, a-series and
p-series of subclones had karyotypes similar to that of the
original BS181 clone. In this case there was no systematic
loss of any rat chromosome from the h- and a-series of
subclones: instead, it was detected that each of these
subclones had undergone a specific chromosomal change:
a deletion of rat chromosome 5. Examples of complete
karyotypes of BS181hl and BS181a3 are given in Figs 5
and 6. In contrast, cells of the non-transformed BS181
subclones of the p-series displayed one or two copies of
rat chromosome 5 with no signs of deletion.
The fact that the five h- and a-subclones isolated
independently from BS181 were all characterized by a
deleted rat chromosome 5 raises the question of whether
the deletion was the same in the different subclones. In
Suppressor gene in rat chromosome 5
149
Table 2. Average number of mouse- and rat-derived chromosomes in the parental cells, and in the mouse-rat cell
hybrids BS140 and BS181 and derived lines
Average number of chromosomes (range)
Parents
BWTG3
RSF
Total
Rat
Mouse
Cell line
42
66-7 (63-70)
42
66-7 (63-70)
Hvbrids
BS140
60-1 (53-67)
56-3 (52-62)
62-0 (59-66)
61-0(56-65)
35-7 (32-40)
95-8 (89-101)
BS140hl
BS140h2
BS140h3
31-7(29-34)
31-1 (29-34)
30-6 (28-35)
88-0 (83-96)
93-1 (90-96)
91-6(88-96)
BS 140a 10
BS140a20
BS140a31
61-8 (60-64)
55-4 (51-62)
57-6 (54-60)
27-6 (26-30)
33-1 (31-35)
29-6 (27-31)
89-4 (87-93)
88-5 (83-94)
87-2 (83-91)
BS181
BS181hl
61-9 (55-67)
36-6(31-41)
98-5 (86-103)
58-6 (53-62)
35-9 (33-39)
BS181a2
BS181a3
BS181a4
BS181a5
56-1
55-5
54-4
52-2
57-9
59-9
320
39-4
36-1
36-6
40-0
38-4
94-5
88-1
94-9
90-5
88-8
97-9
98-3
BS181plO
BS181pll
(48-61)
(53-59)
(46-58)
(44-58)
(41-63)
(51-71)
(26-36)
(36-43)
(32-40)
(35-39)
(30-48)
(34-40)
(90-99)
(74-97)
(91-100)
(84-94)
(80-93)
(71-111)
(90-107)
q22-23 of rat chromosome 5. We propose to call this gene
SAI1 (for suppression of anchorage independence).
It was observed that in the transformed BS140 h- and
a-sublines, the loss of rat chromosome 5 was accompanied by an increase of mouse chromosome 4
(Tables 3 and 5). No corresponding increase was seen in
the non-transformed BS181 p-sublines (which retained
rat chromosome 5) or in the transformed BS181 a- and
p-sublines (which retained a deleted rat chromosome 5).
Obviously, this may be purely fortuitous, but since
mouse chromosome 4 appears to be largely homologous
to rat chromosome 5 on the gene level (see Discussion),
Fig. 7, instances are presented of rat chromosome 5 from
BS181 and the five BS181 h- and a-subclones together
with a diagrammatic representation of a normal rat
chromosome 5. It is evident without any doubt that the
segment deleted in each of the five sublines was the same,
namely band 5q22-23.
The combined information in Tables 1 and 5 makes it
obvious that recovery of anchorage independence in the
hybrids is correlated with loss of chromosomal material
from chromosome 5 of the normal parental rat cell
(Table 6). The inevitable conclusion is that a suppressor
of the transformed phenotype is located in the segment
Table 3. Average number per cell of each individual mouse chromosome in parental BWTG3 cells and in the
mouse-rat cell hvbrids BS140 and BS181 and derived sublines
Mouse chromosome
X
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Marker
1-0
1-9
3-8
2-5
2-0
2-0
4-0
3-1
20
2-9
3-0
4-6
2-7
2-9
2-1
4-7
3-0
3-0
4-7
2-5
8-2
0-9
2-7
2-5
2-8
2-7
3-1
1-8
1-9
4-3
3-6
4-3
3-4
2-7
0-5
0-7
0-8
1-8
1-8
0-9
1-6
1-6
2-4
2-7
4-8
4-0
4-4
2-8
3-1
3-0
30
2-8
2-8
1-0
1-1
1-1
1-0
10
0-9
3-9
5-0
4-8
4-8
3-8
3-8
1-8
0-9
0-7
0-9
0-9
0-8
2-1
3-4
2-0
2-6
21
2-9
1-9
2-2
1-9
1-2
1-2
1-8
1-5
3-2
2-4
2-5
2-5
2-9
1-9
3-0
3-9
4-4
1-6
1-1
1-3
1-1
1-5
1-1
11
0-9
1-2
3-6
3-0
2-4
3-3
3-1
1-0
4-4
4-0
2-2
3-4
3-2
0-6
2-6
BS181plO
BS181pll
0-9
1-0
1-6
1-9
2-0
2-8
2-3
2-8
1-8
1-2
11
11
3-5
4-8
2-6
31
2-0
1-5
2-7
3-0
2-0
2-0
2-3
2-3
2-1
2-0
0-9
1-8
20
3-4
1-8
1-6
1-8
1-8
1-7
1-8
1-0
1-1
3-2
3-4
1-9
1-8
1-2
1-0
3-8
4-3
2-6
2-6
2-8
3-5
2-2
1-8
1-3
1-8
11
1-5
1-2
2-2
2-5
2-1
3-0
2-7
2-9
1-9
2-5
8-8
8-8
13-3
10-5
11-9
9-8
9-7
2-9
2-9
21
2-9
30
2-8
1-0
0-9
2-0
1-9
1-1
2-8
1-9
2-0
2-5
20
3-7
2-8
3-1
1-9
1-9
2-0
0-9
1-7
2-8
1-6
2-4
2-4
1-1
2-6
1-0
2-0
2-0
2-2
2-1
1-8
2-2
2-9
2-2
2-7
30
4-4
3-6
1-5
3-4
2-9
2-7
2-8
2-4
2-1
3-6
4-0
5-0
4-4
4-2
3-2
4-0
3-4
3-4
4-5
4-8
41
3-7
3-2
4-9
4-2
4-5
4-0
4-6
5-0
2-2
4-2
4-8
5-3
1-7
1-2
1-9
1-5
10
10
0-9
BS181
BS181hl
BS181a2
BS181a3
BS181a4
BS181a5
2-6
2-2
2-4
2-3
2-8
1-9
30
3-4
4-2
3-7
3-7
2-9
3-5
2-6
4-5
5-1
4-7
1-7
10
10
1-0
1-1
0-9
1-0
1-0
0-9
1-8
1-0
0-7
0-8
0-7
0-9
0-9
0-4
1-1
0-7
1-1
11
0-9
Cell line
Parent
BWTG3
Hybrids
BS140
BSHOhl
BS140h2
BS140h3
BS140al0
BS140a20
BS140a31
150
1-0
10
11
M. Q. Islam et al.
2-6
11
1-7
2-2
0-7
1-8
2-8
2-7
2-7
2-8
2-6
2-8
3-3
3-0
3-6
3-5
3-6
3-4
12-4
10-6
10-0
7-5
14-3
8-8
12-7
9-2
Table 4. Average number per cell of recurrent mouse-derived marker chromosomes in the parental mouse BWTG3
line and in the hybrids BS140 and BS181 and derived cell lines
Marker chromosome*
Cell line
Parent
BVVTG3
Hybrids
BS140
BSHOhl
BS140h2
BS14Oh3
marl
mar2
mar3
mar4
mar5
mar6
1-0
1-0
1-0
—
—
_
1-0
0-9
0-7
0-7
0-6
3-0
1-6
0-8
1-8
1-8
2-0
0-9
0-8
0-9
10
1-1
1-4
0-8
1-0
1-8
0-1
0-9
0-6
0-9
0-4
1-7
1-2
2-1
1-1
1-5
1-2
0-9
1-0
11
1-0
10
1-0
1-3
0-9
1-0
0-9
1-0
0-6
0-7
BSHOalO
BS140a20
BS140a31
0-9
1-0
0-9
0-8
BS181
BSlSlhl
1-1
-
0-8
0-7
BS181a2
BS181a3
BS181a4
BS181aS
0-7
0-8
0-3
0-1
0-1
BS181plO
BSlSlpll
10
0-7
0-8
10
1-2
1-S
1-2
0-9
0-7
-
mar7
mar9
marlO
01
0-4
0-9
0-7
0-6
1-6
-
0-1
0-2
-
:
Only the most common and clearly identifiable markers have been listed; a number of other markers were also seen sporadically.
*The marker chromosomes listed mostly represent Robertsonian translocations and have the following compositions: marl, rob(l; 1); mar2,
rob(l;8); mar3, 15p+; mar4, rob(10;10); mar5, rob(6;6); mar6, rob(17;17); mar7, rob(8;13); mar8, rob(8;17); mar9, rob(9;17); marlO,
rob(12;12). Actual pictures of these markers can be seen in Figs 1-6.
Table 5. Average number per cell of each individual rat chromosome in mouse-rat hybrids BS140 and BS1SJ and
derived cell lines
Rat chromosome
Hybrid
line
X
Y
-
2-4
1-
2-1
1-8
1-3
1-9
1-9
1-S
—
-
1-0
•1
•6
; •8
; •0
: •8
2-3
2-1
1-4
1-9
10
1-5
2-0
20
1-5
1-6
1-8
1-6
1-1
1-8
>9
1-0
1-8
1-0
1-0
•0*
2-0
1-9
2-1
2-0
1-9
21
1-9
2-0
1-9
2-3
1-8
1-0
2-0
1-9
1-9
2-1
1-9
1-8
0-9
1-6
1-6
2-1
1-0
1-0
0-9
1-1
1-0
1-7
1-9
1-8
1-8
1-7
1-8
2-0
2-0
—
—
()-9
1-9
1-9
1-8
1-0
2-0
10
1-2
0-9
11
1-9
1-8
1-1
1-1
1-8
1-7
\-2
1-7
M
1-9
1-9
3-0
2-0
2-2
2-0
•6
•6
2-6
1-0
1-9
1-9
BS140
2-0
BSHOhl
BS140h2
BS140h3
1-9
2-0
1-9
BSHOalO
BSH0a20
BS140a31
BS181
BS181a2
BS181a3
BS181a4
BS181aS
1-9
1-9
1-9
1-0
1-0
1-0
1-0
1-0
1-0
1-0
1-0
1-0
1-0
1-0
1-0
BS181plO
BS181pll
1-0
1-1
0-7
10
BSlSlhl
•8
1-1
•1
•0*
•0*
•0*
•0*
10
11
12
13
14
15
1-7
1-6
1-2 1-
1-0
11
0-9
1-8
1-7
1-8
1-0
1-0
1-0
1-6
1-3
1-2
1-6
1-2
1-1
1-1
1-3
1-3
20
1-3
1-7
11
1-0
1-0
11
0-9
1-0
2-0
1-0
1-2
1-8
1-0
10
2-5
2-7
2-9
2-4
2-8
1-0
10
1-0
1-9
2-7
1-9
2-9
1-8
2-1
1-9
2-0
2-0
1-9
1-7
1-8
2-1
1-8
1-9
1-0
1-9
1-3
1-3
1-2
10
1-0
1-1
0-9
1-0
2-0
0-5
1-5
1-8
1-8
1-9
1-4
1-9
1-8
1-0
1-8
1-9
1-8
0-9
2-0
2-0
20
1-3
1-2
1-8
1-9
1-6
1-9
2-0
1-7
2-5
1-5
2-0
1-8
20
2-0
21
1-9
1-9
1-8
2-0
1-9
1-6
1-7
1-8
1-9
2-1
16
17
18
19
20
1-5 1-
1-5
2-1
1-9
2-9
2-1
2-2
1-9
1-6
1-3
11
1-3
1-3
1-0
1-8
1-9
1-5
1-8
1-9
1-3
3-5
2-1
1-8
2-4
1-6
1-9
3-2
2-8
1-0
1-8
10
1-6
1-8
1-5
1-8
2-0
2-0
0-8
1-0
11
1-7
0-5
1-4
2-1
2-1
2-1
1-7
11
1-6
1-9
2-0
\-2
1-7
1-7
1-9
10
1-9
1-0
1-7
2-7
1-2
2-6
1-1
1-8
1-9
2-4
1-5
1-9
1-8
2-1
1-9
1-9
1-5
1-6
Rat marker chromosomes have been scored among the normal chromosomes they were derived from.
As mentioned in the text, rat-derived chromosome markers occurred very sparsely. Often they were seen in single cells only (6 instances).
Deletion of the short arm of chromosome 3 was seen occasionally, and (possibly as a consequence of a 3p deletion) Robertsonian translocations
involving both copies of chromosome 3 (5 instances) or chromosome material added at the centromere of chromosome 3(11 instances). None of
the aberrations involving breakage at the centromere of chromosome 3 was seen consistently in any of the lines. Only two cases of rat-derived
translocation chromosomes were seen regularly in the majority of the cells of specific lines: (1) a chromosome derived from a translocation event
involving chromosomes 3 and 4, namely der(4) (4cen-4q22::3ql2-3qter), was present in 9 out of 12 cells analysed from BS181a2 (scored as
chromosome 4 in the Table); (2) in all the subclones of the BS140 h- and a-series each copy of chromosome 11 displayed additional chromosome
material located terminally in the short arm. This material could not be identified unequivocally, but the G-banding pattern (or rather lack of
banding) was consistent with that of the terminal segment of rat chromosome 5 (bands 5q35-qter).
*The number S chromosomes marked by an asterisk display a deletion of the region q22-23.
Suppressor gene in rat chromosome 5
151
Mouse
M
t
21 i» s,<
in isis a
6
7
8
9
W
11
12
13
14
15
W **
4A/f
A£A
i
I
tkv
17
lit
18
19
i
v
16
r r ^ # e
B C
0
E
F
G
H
I
J
K
Rat
!!!! ft M
•
I
15
1« _
_
ft
11
12
•«
Sf
M
17
.__.«_
W_
13
14
««
20
XX
Fig. 1. Karyotype of cell from the anchorage-dependent hybrid BS140. Mouse-derived marker chromosomes: A, rob(l;l);
B, rob(l;8); C, rob(10;10); D, 15p+; E-K, unidentified. Note presence of two normal copies of rat chromosome 5. (The
presence of 4 copies of rat chromosome 1 was only seen in this particular cell.)
152
M. 0. Islam et al.
Mouse
it
a
s
iitft i% 62
Ii
11
12
13
14
16
17
18
19
E
F
G
Ii
15
H
I
J
K
L
M
Rat
! )) n I
•
9
10
11
11
13
I
ii
a
ii
if
.
$f
U
16
,17 .
1*
If
20
XX
.
14
_.M
Fig. 2. Karyotype of cell from the anchorage-independent hybrid subline BS140h3, isolated from cell-dense focus in BS140.
Mouse-derived marker chromosomes: A, rob(l;l); B, rob(l;8); C, rob(10;10) (2 copies); D,E, ISp-l- (2 copies); F, rob(6;6);
G, rob(3;7); H, rob(17;17); I-M, unidentified. Note absence of rat chromosome 5.
Suppressor gene in rat chromosome 5
153
Mouse
It?
3
i
ft*
7
4
13
• ftff
III
t
15
»
«
C
fi
18
17
10
14
12
11
9
19
»
.
.
.
H
Rat
I
I
1
I
8
I
15
n
4
f
10
i
3
Mr
11
18
f
12
14
20
ii
Fig. 3. Karyotype of cell from the anchorage-independent hybrid subline BS140a20, isolated in soft agar from BS140. Mousederived marker chromosomes: A, rob(l;8); B, rob(10;10); C, 15p+; D, rob(6;6); E, rob(8;13); F - K , unidentified. Note
absence of rat chromosome 5. (The translocation between rat chromosomes 1 and 13 was only seen in this particular cell.)
154
M. Q. Islam et al.
Mouse
im
i
•<
i
Hi
I
10
it
I;
13
14
il
15
UM t o
16
It
(
It
X
mlr...
A
B
C D
E
F
G
H
Rat
M II I
))
it it a u
10
11
15
If
n
u
12
I!
17
II
U
13
•
19
If
f
20
U
14
U
X Y
Fig. 4. Karyotype of cell from the anchorage-dependent hybrid BS181. Mouse-derived marker chromosomes; A, rob(l;8);
B, rob(10;10); C, 15p+; D, rob(6;6); E, rob(15;18); F - H , unidentified. Note the presence of a normal copy of rat
chromosome 5.
Suppressor gene in rat chromosome 5
155
Mouse
{
i*i llili *•«» i
4
if
n
;;
?
a
13
u
IH
;W
16
17
1«
H
11
:c
12
I
n
t
X
i I I
H
U
I
It* i»
I
».
:t
••
•
•
10
11
12
13
14
15
IS
17
It
20
X Y
19
Fig. 5. Karyotype of cell from the anchorage-independent hybrid subline BS181hl, isolated from cell-dense focus in BS181.
Mouse-derived marker chromosomes: A, rob(l;8); B, rob(10;10); C, 15p+; D, rob(14;17); E, unidentified; F, rob(7;13);
G-J, unidentified. Note presence of an interstitial deletion in rat chromosome 5.
156
M. 0. Islam et al.
Mouse
t
iti IKS H»
%a
*
»
5
12
13
14
15
l*»
I
11
Mil
16
17
B
C
18
D
E
19
F
JC
6
Rat
J
1
1*
!
2
121
8
i»
15
3
fti
It
1
1
fA
11
12
4
5
9
10
M
ift
1-
11
17
18
It
If
11
6
u
13
20
«%
14
X Y
j
Fig. 6. Karyotype of cell from the anchorage-independent hybrid subline BS181a3, isolated in soft agar from BS181. Mousederived marker chromosomes: A, rob(l;l); B, rob(10;10); C, 15p+; D - G , unidentified. Note presence of interstitial deletion
in rat chromosome 5.
Suppressor gene in rat chromosome 5
157
RN05
BS181
BS181
hi
a2
a4
a3
a5
Fig. 7. Diagram of rat chromosome 5 and cutout instances of rat chromosome S from the anchorage-dependent hybrid BS181
and anchorage-independent sublines, the latter exhibiting the interstitial deletion of bands q22-23.
Table 6. Correlation of loss of chromosome 5 material
from the normal rat parent and anchorage-independent
groivth in the mouse-rat hybrids
Hybrid line
Rat
chromosome 5
Anchorage dependence
BS140
Present
Dependent
BSHOhl
BS140h2
BS140h3
BSHOalO
BS140a20
BS140a31
Absent
Absent
Absent
Independent
Independent
Independent
Absent
Absent
Absent
Independent
Independent
Independent
BS181
Present
Dependent
BSlSlhl
BSl8la2
Deleted
Deleted
Deleted
Deleted
Deleted
Present
Present
Independent
Independent
Independent
Independent
Independent
Dependent
Dependent
BS181a3
BS181a4
BS18la5
BS181plO
BS18lpll
the observed increase in mouse chromosome 4 may be
required to compensate for the loss of some housekeeping
gene functions on rat chromosome 5.
The rat Sc/22-23 region contains the alpha-interferon
locus
The assignment of known genes to the rat 5q22-23 region
would provide 'landmarks' useful, for instance, for
chromosome walking experiments aimed at identifying
the SAI1 gene, or for comparative mapping with the
human genome. Several genes have been assigned by us
(unpublished data) and by others to rat chromosome 5.
Using gene-specific probes in Southern blot analyses, we
have determined whether any of them was included in the
deletion present in the BS181 a- and h-hybrids. Among
the loci tested, a single one, namely the alpha-interferon
locus, was found to map in the 5q22-23 segment.
Dijkema et al. (1984) showed that the rat genome
contains several alpha-interferon genes, among which at
least one, the alphai-interferon gene, is functional; this
gene is contained in a 2-5 kb EcoRl restriction fragment
158
M. 0. Islam et al.
M
R
1
2
3
4
7-
25-
1-1-
*
Fig. 8. Southern blot analysis showing the loss of rat alphainterferon sequences in the transformed BS sublines. The
DNAs were digested with EcoRl and the filter was probed
with the rat alpha!-interferon gene. Lanes: M, mouse DNA
(BWTG3); R, rat DNA; 1, BS181; 2, BSHOhl; 3,
BS181hl; 4, BS181a3; note that the rat 2-5 and 7kb
fragments are lost in the transformed sublines.
of rat genomic DNA. As illustrated in Fig. 8, we found
that this 2-5 kb fragment is lost in the transformed BS140
hybrids of the h- and a-series, which lack rat chromosome
5, and also in the transformed BS181 hybrids of the hand a-series, which possess the deleted rat chromosome
5. Thus, the rat alpha r interferon gene is located in 5q2223. The probe also revealed another rat EcoRl fragment
of about 7 kb, which very probably corresponds to
another alpha-interferon gene. This fragment was also
lost in the transformed hybrid sublines of BS140 and
BS181. Therefore, we conclude that the deletion covers
at least part of the alpha-interferon locus (possibly the
whole locus) and includes the functional alphapinterferon and at least another alpha-interferon gene.
Discussion
The present work was undertaken to study suppression of
the transformed phenotype (anchorage independence) in
interspecific somatic cell hybrids (mouse hepatoma X
normal rat fibroblasts), which turned out to have the
remarkable and extremely favourable property of segregating only a minimum of chromosomes at mitosis. This
unusual mitotic stability of the interspecific BS-hybrids
enabled us to determine unequivocally the identity of
each and every chromosome and to decide easily whether
it came from the transformed or the normal parent. This
fortunate situation led us to the most significant result of
the present study: we established that the anchorage
dependence was under control of a gene (group of genes)
in rat chromosome 5, or more precisely in 5q22-23.
The question arises of whether recovery of many
transformed sublines represents independent events. By
comparing the chromosome composition of each subline
(especially the marker chromosomes) it becomes apparent that there were clear-cut differences in karyotype
make-up among the individual sublines of each primary
hybrid. By itself this is not necessarily evidence for their
independent origin. As is evident from the karyotype data
presented, there is a moderate chromosomal evolution in
these hybrids, and, although the data provide unmistakable evidence that the sublines are quite distinct from
each other, the simplest explanation, in our opinion, is
that they have been derived from subpopulations present
in the hybrids, which could easily have originated from
single deviating cells. This interpretation is supported by
the facts that: (1) the deletion of chromosome 5 present
in the a- and h-sublines of BS181, morphologically
appears to be identical in all of the sublines; and (2) the aand h-sublines of BS140 all carried the same rat-derived
l l p + marker chromosome (Table 5).
A few hybrids (type II hybrids), which had acquired
an excess of mouse hepatoma chromosomes and lost rat
chromosomes, retained the transformed properties of the
parental hepatoma cells. As discussed (Szpirer & Szpirer,
1980), the lack of suppression in these hybrids is probably
due to gene-dosage effects. Indeed, we found (data not
shown) that non-deleted rat chromosome 5 is present in
some such hybrids; consequently, one can suggest either
that the hepatoma chromosome homologous to rat
chromosome 5, namely mouse chromosome 4, carries a
mutated version of the SAI1 gene, encoding an altered
SAI1 product competing with the normal rat SAI1
product, or that other mouse hepatoma chromosomes
contribute genes directing the synthesis of proteins
antagonizing the rat SAI1 product. Such a possibility
might be illustrated by the recently discovered association between oncogene and anti-oncogene (emerogene)
products (Whyte et al. 1988).
Our conclusion about the SAI1 gene in rat chromo-
some 5 must be considered in the light of conclusions
from earlier cases of similar work in the mouse. Jonasson
et al. (1977) reported that in intraspecific mouse hybrids,
malignant X normal, there was a selective pressure both
in vivo and in vitro acting against the copies of chromosome 4 derived from the normal parent. They concluded
that the locus against which the selective pressure was
directed must be 'in the lower part of the upper half of
mouse chromosome 4. Again, Evans et al. (1982) confirmed that in similar crosses the suppression of tumorigenicity emanated from chromosome 4 of the normal
parent.
Several workers have pointed out that the G-banding
pattern of rat chromosome 5 is very similar to that of
mouse chromosome 4. Gene assignments in the rat
determined by other workers and by ourselves (for
reviews, see Levan et al. 1986; O'Brien, 1987; Szpirer
et al. 1988) have shown that the gene contents of rat
chromosome 5 and mouse chromosome 4 exhibit a
considerable degree of homology. The similarity in
morphology of these two chromosomes is evident
(Fig. 9). We have tried to interpret the information in the
paper by Jonasson et al. (1977) in terms of the banding
nomenclature of Nesbitt & Francke (1973) and have
indicated to the right of the figure the approximate limits
of the chromosome segment that Jonasson et al. identified as being counterselected against. Assuming the gene
homology between mouse chromosome 4 and rat chromosome 5 is reflected by their morphological homology, the
deletion found by us in rat chromosome 5 of the nonsuppressed BS181 sublines (a- and h-series) corresponds
to part of the segment of mouse chromosome 4 implicated
in the study of Jonasson et al. Hence, it seems quite
possible that the SAI1 gene in rat chromosome 5 is
homologous to the mouse suppressor gene detected by
Jonasson et al. (1977).
Rat chromosome 5 (and mouse chromosome 4) share
gene homology with human chromosomes 1 (the short
arm; lp) and 9 (see Searle et al. 1987). Consequently, it
is extremely exciting that the presence of a suppressor
gene, acting both /// vitro and in vivo, located in the short
arm of human chromosome 1 has been indicated in
several lines of inquiry, including the following: (1) in
somatic cell hybrids between malignant Syrian hamster
cells and normal human fibroblasts, anchorage dependence was correlated with the presence of the short arm of
human chromosome 1 (Stoler & Bouck, 1985). (2) In
intraspecific hybrids between human sarcoma cells and
normal human fibroblasts, retention of chromosome 1
was apparently correlated with suppression of the malignant phenotype (Benedict et al. 1984). (3) Deletions
and/or rearrangements of the terminal portion of lp
(Ip31-lpter) are very common in human neuroblastomas
(Brodeur et al. 1981; Gilbert et al. 1982; Kaneko et al.
1985), indicating the presence of an emerogene in this
segment. (4) Finally, in a search for loss of heterozygosity
in tumour DNA from 15 blood/tumour pairs in patients
with the dominantly inherited cancer syndrome multiple
endocrine neoplasia type II, Mathew et al. (1987)
demonstrated loss of heterozygosity for a probe specific
for the short arm of chromosome 1 in seven out of 14
Suppressor gene in rat chromosome 5
159
informative pairs. The chromosomal specificity of the
loss was tested on the same filters with probes from five
other chromosomes and loss of heterozygosity was observed in no case. Heterozygosity was also conserved for a
probe specific for the long arm of chromosome 1. Thus,
the SAI1 gene in rat chromosome S might be homologous
to an emerogene located in human chromosome 1.
The only locus, however, that we could assign to the
rat 5q22-23 region was the alpha-interferon locus. Obviously, the presence of this locus in the chromosome
region containing the SAI1 gene may be fortuitous and
the two loci may just be closely linked. A rough estimate
of the size of the deletion gave the approximate value of
10 or 20 megabases; a region of that size might harbour
many genes. Still, the possibility remains that the SAI1
gene in fact is an alpha-interferon gene. It is well known
that interferons possess growth-inhibiting properties
(Taylor-Papadimitriou, 1984). In any case, this finding
puts the human chromosome 9 into focus, since the
alpha-interferon maps to 9p22-13 in man (Trent et al.
1982). In early reports, evidence from somatic cell
hybrids was presented indicating that human chromosome 9 would be involved in tumour suppression
(Klinger et al. 1978; Klinger, 1982). We have found that
in hybrids between mouse L cells and human fibroblasts,
which segregate human chromosomes, the human
chromosome 9 is lost preferentially (Wathelet et al. 1988;
and our unpublished observations), possibly indicating
that hybrids containing human chromosome 9 are at a
growth disadvantage. A similar finding has been reported
by Ruddle & Creagan (1975).
Even stronger indications that human chromosome 9
contains an emerogene can be found by a systematic
search for specific chromosome changes in human tumours. A computer printout from the database on
chromosome aberrations in human cancer (see Mitelman,
1988) showed that bands 9p22-13 were often involved in
deletions or translocations. The overall frequency of
2-4% among all 9029 individual cases present in the
registry might not sound so impressive, but in some
specific types of malignancies the frequency was quite
substantial. Thus, deletions or translocations in bands
ft
5q22
5q23
4A4
4C3
RNO5
MMU4
Fig. 9. Diagrams and actual instances of rat chromosome 5
(left) and mouse chromosome 4, showing the morphological
similarity between the two chromosomes. On the far left the
extent of the deletion in the anchorage-independent sublines
of BS181 has been indicated. On the far right we have
indicated (in terms of the terminology of Nesbitt & Francke,
1973) the extreme limits for the segment including an
emerogene detected by Jonasson et al. (1977) in the mouse.
9p22-13 were found in 70 cases (8-5 %) of acute lymphocytic leukaemias. On the basis of the prevalence of 9p
aberrations in this disease, Chilcote et al. (1985) actually
suggested that an emerogene must be present in this
region. Furthermore, a t(9; 11) translocation involving
bands 9p22-13 was very common (13-7%) in acute
monocytic leukaemia and in about half of the cases it
represented the only aberration detected. Finally, in
some solid tumours deletions and translocations in 9p2213 were common (e.g. in more than 50% of astrocytomas). A compilation of the findings is presented in
Table 7. Last but not least, homozygous deletions of the
alpha- and beta-interferon genes have recently been
discovered in human leukaemia cell lines (Diaz et al.
1988). Taken together, these data strongly indicate that
an emerogene may be located in the short arm of human
chromosome 9. This emerogene may well be homologous
to our rat SAI1 gene.
Access to the Registry of Chromosome Aberrations in Human Neoplasms was generously provided by Professor Felix
Mitelman, Lund. We thank Michele Riviere for technical
assistance and Ton Kos, TNO Primate Center, Rijswijk,
Holland, for the gift of pPCl. This work was supported by
grants from the Swedish Cancer Society, the Erik PhilipSorensen Foundation, the Nilsson-Ehle Foundation and CAN-
Table 7. Cases of human neoplastic disease with involvement of chromosome breakage at bands 9p22-13
Number of cases with involvement of9p22-13
Overall frequency
*i m / ^ t i fr o i l
r"i
Cfc
dlllOIIg all Ld&eb
Disease group
Acute non-lymphocytic leukaemia
Deletion
Translocation
6(1)
1
44 (22)
Myeloproliferative disorders
Myeiodysplastic syndromes
Acute lymphocytic leukaemia
Chronic lymphoproliferative disorders
2(2)
41(7)
2
Malignant lymphomas
12
Epithelial neoplasms
Mesenchymal neoplasms
Melanocytic neoplasms
Malignant neurogenic neoplasms
4
1
2
14
5
1
29(6)
9(1)
18
15(2)
4
2
8
Total
registered (%)
50 (23)
2-4
6
3(2)
70(13)
11(1)
0-3
0-3
8-5
30
19(2)
5
4
22
1-8
2-9
2-4
2-6
6-9
9-8
Data derived from the Registry of Chromosome Aberrations in Human Cancer (Mitelman, 1988). Numbers in parenthesis refer to cases
where an aberration involving 9p22-13 was the only aberration detected in the tumour cells.
160
M. Q. Islam et al.
CIRCO (Gothenburg), and the CGER-ASLK, the Belgian
Ministere de la Politique Scientifique, and the ASCC-SVTK
(Brussels). C. S. and J.-F. D. are senior research associate and
research assistant, respectively, of the National Fund for
Scientific Research (Belgium).
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DIAZ, M. O., ZIEMIN, S., L E BEAU, M. M., PITHA, P., SMITH, S.
D., CHILCOTE, R. R. & ROWLEY, J. (1988). Homozygous deletion
of the a- and ^pinterferon genes in human leukemia and derived
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(Received 2 August I98S -Accepted
7 October 1988)