Cell, Vol. 32, 1171-I
0092-8674/83/041171-10
160, April 1983 0 1983 by MIT
$02.00/O
Cytoplasmic Activation of Human
Nuclear Genes in Stable Heterocaryons
Helen M. Blau,* Choy-Pik Chiu and
Cecelia Webster
Department
of Pharmacology
Stanford University School of Medicine
Stanford, California 94305
Summary
We have induced the stable expression
of musclespecific genes in human nonmuscle
cells. Normal
diploid human amniocytes
were fused with differentiated
mouse muscle cells by using polyethylene
glycol. The fusion product, a stable heterocaryon
in
which the parental cell nuclei remained
distinct, did
not undergo division and retained a full complement
of chromosomes.
This is in contrast
with typical
interspecific
hybrids (syncaryons),
in which the parental nuclei are combined
and chromosomes
are
progressively
lost during cell division.
The human
muscle proteins,
myosin light chains 1 and 2, MB
and MM creatine
kinase and a functional
mousehuman hybrid MM enzyme molecule were detected
in the heterocaryons.
Synthesis
of these proteins
was evident 24 hr after fusion and increased
in a
time-dependent
manner thereafter.
Our results indicate that differentiated
mouse muscle nuclei can
activate human muscle genes in the nuclei of a cell
type in which they are not normally expressed,
and
that this activation
occurs via the cytoplasm.
The
activators
are still present in cells which have already initiated
differentiation,
are recognized
by
nuclei of another
species, and do not diffuse between unfused cells. The reprogrammed
amniocyte
nuclei of stable heterocaryons
provide
a unique
system in which to study the mechanisms
regulating
gene expression
during cell specialization.
Introduction
Muscle cells provide
an advantageous
system in
which to examine biochemical
and molecular changes
associated with differentiation.
Mononucleated
myoblasts spontaneously
fuse to form multinucleated
myotubes, which become visibly striated and rhythmically
contract in tissue culture. These distinct morphological changes
are accompanied
by the cessation
of
DNA synthesis and the initiation of synthesis of several
muscle enzymes, structural proteins of the contractile
apparatus
and specialized
membrane
receptors
(Shainberg
et al., 1971; Perriard et al., 1978; Garrels
and Gibson, 1978; Devlin and Emerson, 1979; Devreotes et al., 1977; Weinberg et al., 1981; for review,
see Pearson, 1980). Although this sequence
of biological events has been well characterized,
the regulatory mechanisms
underlying
the transition
to the
* To whom
reprint
requests
should
be addressed.
differentiated
state remain poorly understood.
In addition, no muscle stem cell, or undetermined
myoblast
precursor, has yet been identified in which to examine
the initial commitment to cell specialization.
Studies of differentiation
using somatic cell hybrids,
or syncaryons,
have demonstrated
the existence of
regulatory
factors capable of repressing
or inducing
the expression
of differentiated
functions (Ephrussi,
1972; Davidson and de la Cruz, 1974; Davis and
Adelberg,
1973; Ringertz and Savage, 1976; Weiss,
1977). Extinction of a differentiated
protein was typically observed
upon fusion of a cell that expressed
the protein with a cell that did not (Davidson et al.,
1966; Davidson
and Benda, 1970; Bertolotti
and
Weiss, 1972; Mevel-Ninio
and Weiss, 1981). Evidence that extinction was due to the product of a gene
encoding
a repressor was best demonstrated
by the
re-expression
of the extinguished
function following
chromosome
loss (Klebe et al., 1970; Weiss and
Chaplain, 1971). Activation of a gene was occasionally observed when the genome of the differentiated
cell type was present in increased dosage (Peterson
and Weiss, 1972; Darlington et al., 1974; Brown and
Weiss, 1975; Darlington
et al., 1982). During the
propagation
of such interspecific
hybrid cells, however, chromosomes
were frequently
lost so that the
genetic composition
of the hybrids was unstable and
highly variable. As a result, syncaryons
became invaluable for gene mapping, but were of limited utility
for controlled
studies of gene regulation.
We have developed
a stable heterocaryon
system
that overcomes
many of the problems experienced
with syncaryons
and is ideal for characterizing
the
role of regulatory
factors in cell differentiation.
Our
approach takes advantage
of the fact that the differentiated muscle cell is naturally a nonreplicating
multinucleated cell. Thus, after fusion of muscle with other
cell types, the parental cell nuclei do not divide, but
remain distinct and intact, with the entire chromosome
complement
of each present. Previous attempts to
induce novel gene expression
in heterocaryons
have
failed (Carlsson et al., 1970, 1974a, 1974b; Zeuthen
et al., 1976; Mevel-Ninio
and Weiss, 1981). Here we
demonstrate
that heterocaryons
between mouse myotubes (differentiated
muscle cells) and human amniotic cells (nonmuscle
cells) can be produced
with
high efficiency and that in these stable fusion products, silent human genes encoding muscle M-creatine
kinase and three myosin light chains are activated.
The induction of gene expression
in these heterocaryons demonstrates
conclusively
that factors capable
of activating genes are not limited to the nucleus, but
also exist in the cell cytoplasm.
These factors act
rapidly and can gain access to intact foreign nuclei.
Heterocaryons
of this kind provide a stable and reproducible model system in which to study biochemical
and molecular aspects of regulation
of gene expression during cell specialization.
Cell
1172
Results
Production
of Heterocaryons
The mouse muscle cells used were a permanent cell
line, which predictably
differentiated
to form contractile fibers in culture (Figure 1 a). The human nonmuscle
cells were normal diploid amniotic fluid cells of the F,
Figure
1.
Formation
of Heterocaryons
by Fusion
of Mouse
Muscle
or fibroblastic,
cell type (Figure 1 b). When more than
90% of the muscle nuclei were contained
in actively
contracting
myotubes,
sufficient
amniocytes
were
added to achieve confluence
(Figure 1 c). Cells were
fused by using polyethylene
glycol according
to the
method of Davidson et al. (1976). Unfused cells were
eliminated by treatment of the cultures with selective
Cells and Human
Amniocytes
with PEG
Phase contrast
micrographs
of cells in viva. Mouse myotubes
(a), human amniocytes
(b), confluent
coculture
of myotubes
prior to PEG treatment
cc), and the same culture 6 days after fusion with PEG and treatment
with selective
agents(d).
450x.
and amniocytes
just
Gene Activation
in Stable
Heterocaryons
1173
agents (Figure 1 d). Cytosine arabinoside,
which inhibits DNA synthesis (Cohen, 1966; Yeoh and Holtzer,
1977), was used to eliminate unfused proliferating
myoblasts.
Residual
amniocytes
were removed
by
using ouabain (1 O-5 M), an inhibitor of sodium potassium-ATPase
with a 1 OOO-fold greater affinity for the
human than for the rodent enzyme (Thompson
and
Baker, 1973). The resulting mixture of multinucleated
muscle-amniocyte
heterocaryons
and muscle homocaryons survived a minimum of 6 to 7 days, were
striated and exhibited typical muscle contractile
behavior.
To distinguish
heterocaryons
from homocaryons,
a
means for identifying
the nuclei of the two parental
cell types was required.
For this purpose,
speciesspecific
differences
were used to advantage.
As
shown in Figure 2, the nuclear composition
of heterocaryons formed between cells of mouse and human
was easily determined
by using Hoechst 33258. This
compound
fluoresces
with greater intensity in the
presence of DNA with a high content of adenine and
thymine (Weisblum
and Haenssler,
1974). Satellite
DNA in the centromeric
regions of mouse chromosomes is rich in poly(dAT),
and mouse interphase
nuclei stained with the fluorochrome
appear punctate.
On the other hand, human nuclei, whose DNA does
not contain AT-rich regions, are characterized
by a
generalized
low level fluorescence
(Moser et al.,
1975). This method for identifying nuclei is rapid and
unambiguous.
Efficiency
of Heterocaryon
Production
The efficiency
of heterocaryon
production
and the
average ratio of mouse to human nuclei contained
in
heterocaryons
were determined
by visually scoring
the nuclei stained by Hoechst 33258. The results of
a sample experiment,
in which heterocaryons
were
followed as a function of time after fusion, are shown
in Table 1. Column 2 shows that at each time point,
an average of 73% of all multinucleated
myotubes
were heterocaryons,
or contained a mixture of mouse
and human nuclei. Thus only one quarter of the myotubes present were homocaryons,
composed solely of
mouse muscle nuclei. Thirty heterocaryons
at each
time point were randomly selected for detailed analysis. Heterocaryon
size (column 3), or the total number
of nuclei per heterocaryon,
ranged from 7 to 15, but
did not appear to increase systematically
with time
after fusion. The proportion
of human nuclei in heterocaryons shown as percentage
of human nuclei (column 4) and as the ratio of mouse to human nuclei
(column 5) also remained constant over this period of
time. These results were confirmed
in five separate
experiments
in which heterocaryons
were analyzed
on a daily basis. They suggest that after PEG treatment, heterocaryon
composition
remains relatively
constant, that myotubes do not fuse with one another
and that the fusion of any residual myoblasts to existing myotubes is minimal, if it occurs at all.
Figure
33258
2. Mouse-Human
Heterocaryons
Stained
with
Hoechst
Multinucleated
myotubes
are shown in phase contrast (top) and by
fluorescence
(bottom). Punctate mouse and uniformly stained human
nuclei are clearly distinguished
by the Hoechst stain. 63x.
Synthesis of Human Muscle Proteins by
Heterocaryons
The data presented below represent the results of 18
fusion experiments
in which multiple replicate dishes
were analyzed at several different time points. The
amniocytes
used in these experiments
were fibroblastic F amniocytes isolated from three sources. All
gave consistent and highly reproducible
results.
Creatine Kinase
Creatine kinase(CK)
is of central importance
in generating the energy required
for vertebrate
muscle
contraction.
The enzyme is a homodimer or heterodimer of two subunits, B and M; consequently,
three
isozymes arepossible-BB,
MB and MM. The synthe-
Cell
1174
Table
1. Heterocaryons
Efficiency
of Mouse
Muscle
Cells and Human
Amniocytes
Nuclear
of Fusion”
Compositionb
(1)
C-2)
(3)
(4)
(5)
Days after
PEG Fusion
% Heterocaryons
per Total Myotubes
No. Nuclei
per Heterocaryon
% Human per
Total Nuclei”
Mouse/Human
Nuclei
1
67
7t1
35 rt 3 (176)
2.2
28 f 3(412)
2.8
2
70
3
73
14 f 2
31 f 3 (404)
2.3
4
74
15 f 2
33 f 3 (468)
2.0
5
73
36 +- 3 (249)
2.2
6
80
Mean
73 f
14 f 3
8+1
2
10 2 1
39 + 3 (288)
1.8
11 kl
34 f
2.2
a On each day, 40 myotubes were randomly selected and scored for nuclear
‘Thirty
heterocaryons
were scored each day for the total number of mouse
were human. Data are presented
as the mean f SEM.
‘The total number of nuclei scored each day is indicated in parentheses.
heterogeneity.
and human nuclei
2
in each and for the proportion
of total nuclei
that
MM
MB
BB
Figure
3. Expression
of Human
M-CK
in Mouse-Human
Heterocaryons
Whole cell extracts were subjected
to electrophoresis
on nondenaturing
polyacrylamide
gels for 4 hr (A) and 7 hr (6). CK isozymes (BB, MB and
MM) were detected with UV illumination using a coupled enzyme reaction with NADPH as its end product. (A) lsozymes
from amniocytes
alone
(lane 1); amniocytes
plus PEG (lane 2); cultured mouse muscle (lane 3); cultured human muscle (lane 4); and cultured mouse and human muscle
extracts
mixed in vitro (lane 5). (B) lsozymes
from cultured mouse muscle and human muscle mixed in vitro (lane 1); coculture
of mouse muscle
and human amniocytes
after 6 days without PEG (lane 2); heterocaryons
of mouse muscle and human amniocytes
at the following time intervals
after PEG fusion-l
day (lane 3), 3 days (lane 4) 4 days (lane 5) 5 days (lane 6); 6 days (lane 7); heterocaryons
of mouse muscle and human
muscle formed spontaneously
(lane 8). Arrows indicate CK isozymes containing human subunits.
sis of these isozymes changes with muscle differentiation. BB is the brain or embryonic form and is present
in a number of cell types and in small amounts in
cultured myoblasts.
M subunit synthesis is initiated
following the spontaneous
fusion of myoblasts to form
myotubes. Thus in cultured muscle the MB isozyme is
characteristic
of early stages of muscle differentiation
and is succeeded
by the MM isozyme.
The three isozymes of CK are well separated
by
electrophoresis
of whole cell extracts on nondenaturing polyacrylamide
gels, as shown in Figure 3. The
BB, MB and MM isozymes can be specifically
visual-
Gene
1175
Activation
in Stable
Heterocaryons
ized with ultraviolet illumination
using a coupled enzyme reaction with NADPH as its end product. Myokinase (adenylate
kinase), which frequently
complicates the analysis of CK activity in other gel systems,
does not comigrate with the CK isozymes under our
electrophoretic
conditions,
and has been specifically
inhibited
by the presence
of p’p5-dkadenosine-5’)
pentaphosphate
in the reaction mixture (Lienhard and
Secemski,
1973). The limit of detection
is 1 mU of
enzyme activity, that produced
by approximately
1 O3
well differentiated
mouse muscle cells. As shown in
Figure 3A, human and mouse CK isozymes can be
readily distinguished
by this method (lanes 3 and 4).
Differences
in the mobility of both the MM and MB
isozymes of the two species are especially clear when
mouse and human muscle extracts are combined (lane
5).
Human muscle CK is not normally synthesized
by
either amniocytes or mouse muscle cells. Human amniocytes, like undifferentiated
myoblasts (Blau et al.,
1982, 1983) contain only a small amount of CK, all
of which is the BB isozyme.There
is no evidence of M
subunitsynthesis
in amniocytes alone (Figure 3A, lane
1) or in amniocyte homocaryons
formed by treatment
of the cells with PEG (lane 2). Homocaryons
of C2
muscle cells produced with PEG and exposed to the
selective agents cytosine arabinoside
and ouabain
also do not synthesize an enzyme with the electrophoretie mobility of the human MM isozyme (data not
shown). Thus neither the fusion process per se nor
exposure to the selective agents results in altered
synthesisof
this enzyme by either parental cell type
alone.
The expression
of creatine kinase by amniocytes
and mouse muscle cells fused in heterocaryons
was
examined at time intervals after PEG treatment (Figure
38). For accurate comparison,
cell extracts containing
equivalent
CK activities were layered on gels and
subjected
to electrophoresis.
Within 1 day after fusion, novel isozymes are detectable
in heterocaryons
(lane 3). After 3 days, human MB-CK and a band
intermediate
between mouse and human MM-CK are
clearly evident (lane 4). The intensity of these two
bands continues to increase with time (lanes 4 to 6).
By day 6, a third isozyme, which comigrates with pure
human MM-CK, is distinguishable
(lane 7). By contrast, when mouse muscle cells and amniocytes
not
treated with PEG are grown together for 6 days the
pattern of CK isozymes resembles that of mouse muscle alone (lane 2).
We have identified the intermediate
band between
human MM-CK and mouse MM-CK as a functional
mouse-human
hybrid enzyme by examining
the isozyme composition
in interspecific
muscle heterocaryons. Human muscle cells and mouse muscle cells
plated together in culture spontaneously
fused in the
absence of PEG to form multinucleated
contracting
myotubes. The heterocaryon
nature of the myotubes
was verified with Hoechst 33258, and the average
ratio of mouse to human nuclei determined
to be 2:l.
Like the synthetic muscle-amniocyte
heterocaryons
described
above, the muscle-muscle
heterocaryons
contained
isozymes that comigrated
with the mouse
and human MM standards and a third form intermediate between the two (Figure 3B, lane 8). This hybrid
enzyme was present only in synthetic
or spontaneously formed heterocaryons
in which the cells
shared a common cytoplasm.
It was not present in
mouse and human muscle cell extracts mixed in vitro
(lane 1) or in cocultures of mouse muscle and human
amniocytes
not exposed to PEG (lane 2). This interspecific hybrid molecule was a functional
enzyme,
since the method for visualization
was based on enzyme activity. Although the ratios of mouse to human
nuclei of both the synthetic and spontaneous
heterocaryons were similar, the relative intensity of the human MM-CK band differs between lanes 7 and 8. This
difference is likely to be due to the presence of human
muscle homocaryons
in the spontaneously
fused cultures (lane 8) which contributed
pure human MM
isozyme.
An analysis of nuclear ratios and MM-CK isozymes
in amniocyte-muscle
heterocaryons
reveals that the
efficiency of gene activation is high. Since residual
mouse myoblasts and human amniocytes synthesize
only BB-CK, their presence does not influence the
analysis. The only other source of MM-CK in these
experiments
is homocaryon
mouse myotubes, which
comprise approximately
one quarter of the total myotubes (Table 1, column 2). As shown in columns 4 and
5, one third of the nuclei in heterocaryons
were human. Thus one quarter of all nuclei in myotubes (homocaryons and heterocaryons)
were human, and the
relative contribution
of human M subunits to the observed MM isozymes approximates
this proportion
(lane 7). Within the limits of the assay, these results
suggest that almost every amniocyte
nucleus incorporated into a myotube is activated.
Myosin Light Chains
The myosin light chains, together with the myosin
heavy chains, interact with actin to form the major
components
of the muscle contractile
machinery.
Myosin light chains are not synthesized
by cultured
myoblasts, but appear once muscle differentiation
is
initiated. We partially purified contractile proteins from
differentiated
cultures of human and mouse muscle,
fractionated
a mixture of the proteins of both species
on two-dimensional
gels and visualized the accumulated proteins by staining with Coomassie brilliant blue
(Figure 4). The actins, tropomyosins
and myosin light
chain 3f of the two species comigrated
and could not
be distinguished
by this method, but four of the myosin
light chains were distinct. The first fast myosin light
chains (1 f) differ in molecular weight, the second fast
and slow myosin light chains (2s and 2f) differ in
isoelectric
points, and the first slow myosin light
Cell
1176
these novel proteins was detected 2 days after fusion
and reached a peak on day 4. The gene for the other
myosin light chain (1 f) may also have been activated,
but the protein product was not present in sufficient
amount to be detected by this assay. This result is not
unexpected,
given the differential
synthesis of the
myosin light chains in cultured
mouse and human
muscle cells (see Figure 4). The human myosin light
chains (1 s and 2f) most readily detected in the heterocaryons are those present in greatest amounts in
human muscle cultures.
Discussion
Figure 4. Contractile
cle Cells
Proteins
from Cultured
Mouse
and Human
Mus-
Partially purified proteins from mouse and human cultures ware mixed
in vitro, fractionated
on two-dimensional
gels (O’Farrell,
1975) and
visualized
by staining with Coomassie
brilliant blue. Actins (A) and
tropomyosins
CTm) of the two species comigrate,
but the myosin light
chains Is, If, 2s and 2f of the two species are distinct. Arrows
designate
human (+) and mouse (t) myosin light chains.
chains (Is) differ in both parameters.
The identity of
these proteins was confirmed by using a monoclonal
antibody that recognizes
a determinant
on myosin
light chains 1 and 2 of both mouse and human. Contractile proteins of mouse muscle and human muscle
cultures were separated on two-dimensional
gels, and
the myosin light chains were detected on immunoblots
according to the method of Burnette (1981) as shown
in Figure 5, left. Four of the myosin light chains exhibit
species-specific
differences
and are useful markers
of human muscle gene expression
in heterocaryons.
To determine
whether human myosin light chain
genes were activated in heterocaryons,
we examined
the proteins synthesized in amniocytes, in mouse muscle cells and in heterocaryons
made between the two
cell types (Figure 5, right). Cultures of these cells
were incubated with ?-methionine,
and the labeled
contractile
proteins were partially purified and fractionated on two-dimensional
gels. Autoradiograms
of
the gels revealed that in heterocaryons
(Figure 5,
right, bottom), human myosin light chains 1s and 2f
are synthesized
in substantial amounts, 2s is present
in trace amounts and If is not detectable.
None of
these proteins is evident in either parental cell type
alone (top and middle). To ensure precipitation
of
myosin light chains that might be present in minor
amounts, the labeled amniocyte
proteins were extracted together
with unlabeled
carrier mouse contractile proteins. Mouse muscle cells treated with PEG
and grown in the presence
of cytosine arabinoside
and ouabain also did not synthesize
human myosin
light chains (data not shown).
These data demonstrate
that synthesis of three of
the human myosin light chains is induced following
fusion of amniocytes
with muscle. The synthesis of
One approach to the study of muscle cell determination and the mechanisms underlying cell specialization
is to induce muscle gene expression
in a nonmuscle
cell. We have achieved this by introducing
normal
diploid amniocytes
into the cytoplasm of a differentiated muscle cell. Mouse muscle cells were fused
with human amniocytes to form multinucleated
heterocaryons in which four previously silent genes were
activated
and distinct human muscle proteins detected in amounts typical of human cultured muscle.
Three of these were structural proteins of the contractile apparatus, myosin light chains, and the fourth was
a subunit of a cytoplasmic
enzyme, creatine kinase.
None of these proteins is normally synthesized
by
amniocytes.
To our knowledge
this is the first time
muscle gene expression
has been induced in a nonmuscle cell by cell hybridization.
It is also the first time
gene activation has been observed in a heterocaryon.
The successful activation of previously silent genes
in our heterocaryons
may have been due to the cell
types used. The majority of cell hybridization
experiments have employed cell lines derived from tumors
that were transformed,
highly aneuploid and probably
not optimal for the study of normal gene regulation in
heterocaryons
(see Weiss, 1977, for review). In the
present study, muscle gene expression
was induced
in normal diploid human amniocytes by a diploid myogenic cell line. In addition, the increased gene dosage and karyotypic
stability provided by the natural
multinucleated
state of the differentiated
muscle cell
were used to advantage.
Amniotic fluid cells were
selected as the cell type for induction for two reasons.
First, studies by Darlington
et al. (1982) in hybrids
showed that gene activation occurred with the highest
frequency in this cell type. Second, a reliable method
for activating specific differentiated
gene products in
amniotic fluid cells could provide a novel approach to
the prenatal diagnosis
of human genetic diseases
currently not amenable to detection. Toward this end,
the efficiency and rapidity of gene activation in our
muscle heterocaryons,
which is greater than that observed in most hybrids, is particularly
advantageous.
The muscle-amniocyte
heterocaryon
system we
have developed
is particularly
advantageous
for stud-
Gene
1177
Figure
Activation
in Stable
5. Expression
Heterocaryons
.
of Human
Myosin
Light Chains
in Mouse-Human
Heterocaryons
(Left) identification
of myosin light chains on immunoblots.
The region of the gel in the inset of Figure 4 is shown for cultured human muscle (top),
cultured mouse muscle (middle) and cultured mouse and human muscle mixed in vitro (bottom). Contractile
proteins were partially purified from
differentiated
cultured muscle cells and electrophoresed
on two-dimensional
gels. Myosin light chains of human and mouse species were identified
on Western
blots with a monoclonal
antibody to myosin light chains and a second antibody coupled to biotin and reacted with an avidin-biotin
horseradish
peroxidase.
(Right) Myosin light chain synthesis
in heterocaryons.
Human amniocytes
plus PEG (top); mouse muscle cells plus PEG
(middle); heterocaryons
4 days after fusion with PEG (bottom). Cultured cells were labeled with %-methionine,
and the contractile
proteins were
partially purified, fractionated
on two-dimensional
gels and processed
for autoradiography.
Proteins were identified by comigration
with unlabeled
contractile
protein standards.
Amniocyte
proteins were coextracted
with unlabeled mouse muscle proteins to ensure precipitation
of contractile
proteins. Mouse muscle cells and heterocaryons
were grown in the presence
of ouabain and cytosine arabinoside.
Electrophoresis
of gels at left
and right was performed
at separate times. The positions of the human myosin light chains are indicated by arrows. Myosin light chains Is and 2f
are clearly induced in heterocaryons;
a trace of 2s is also evident.
ies of gene regulation
during differentiation
because
it is stable. Gene activation has previously been observed in syncaryons
(Peterson
and Weiss, 1972;
Darlington
et al., 1974, 1982; Brown and Weiss,
1975) and in cybrids formed by the fusion of the
cytoplasm of one cell type to another intact cell (Gopalakrishnan
et al., 1977; Gopalakrishnan
and Anderson, 1979; Giguere and Morais, 1981; Kahn et al.,
1981 a, 1981 b; Clark and Shay, 1982). However,
gene activation was almost always transient in these
systems because of the loss of nuclear chromosomes
typical of hybrids (syncaryons),
or because of the
dilution and turnover of donor cytoplasmic
proteins
typical of cybrids. Heterocaryons,
on the other hand,
stably retain all nuclear and cytoplasmic
components
of the cell types fused.
Another advantage of the heterocaryon
system we
have developed
is that gene expression
can be examined immediately
after fusion. Numerous cell divisions are usually required to select for and identify
hybrids or cybrids and to obtain sufficient numbers of
these cells for analysis of gene products. By contrast,
heterocaryons
can be identified histochemically
at any
time after fusion. In our experiments,
more than two
thirds of all multinucleated
myotubes consistently contained nuclei of both species. This high efficiency of
heterocaryon
production
permitted
detection
of a
novel gene product in cell extracts 24 hr after fusion.
In addition, the frequency of amniocyte activation was
high: when one quarter of all nuclei in myotubes were
of amniocyte origin, a comparable
proportion of the M
subunits present in the CK isozymes of heterocaryons
were human (Figure 3, lane 6). We therefore think that
the time course and efficiency of gene activation in
Cell
1178
this system is favorable for an attempt to identify the
regulatory factors involved.
In all cases in which we observed activation, muscle
cell nuclei were present in excess. Similar gene dosage effects have been reported
in other systems,
suggesting that an alteration in the balance of repressor and activator regulatory
molecules can influence
gene expression (Peterson and Weiss, 1972; Darlington et al., 1974, 1982; Brown and Weiss, 1975).
Apparently,
these regulatory
molecules
are not restricted to the nucleus, but exist also in the cell cytoplasm. This is clear from the fact that in our heterocaryons, activation of gene expression
was possible,
although
the nuclei of the two cell types remained
distinct.
Moreover,
since the heterocaryons
were
formed by fusing amniocytes with myotubes, the activator(s) were still present in muscle cells in which
differentiation
was already under way. The continued
presence of activator molecule(s) suggests that they
may be required not only to initiate differentiation
but
also to maintain the differentiated
state.
In summary, the reprogrammed
amniocyte of the
muscle heterocaryon
provides a novel approach
to
the examination
of gene regulation
during cell differentiation. The extent to which the expressed potential
of a determined
cell is heritable and stable as a result
of changes in the genome itself and the extent to
which it is reversible
and subject to regulation
by
cytoplasmic factors can be usefully explored in stable
heterocaryons.
This system should be helpful in determining the mechanisms and characterizing
the molecules responsible
for induction and maintenance
of
the differentiated
state in mammalian cells.
Experimental Procedures
Cell Types, Culture and Fusion Conditions
Muscle cells were a diploid continuous
cell line (C2Cj2) originally
isolated by Yaffe from the thigh muscle of a 2 month old C3H mouse
(Yaffe and Saxel, 1977) and subcloned
and karyotyped
in our laboratory. C2C12 cells are maintained as undifferentiated
myoblasts
in a
nutrient-rich
growth medium containing Dulbecco’s
modified Eagle’s
medium (DMEM) supplemented
with 20% fetal calf serum and 0.5%
chicken embryo extract. To induce myoblast fusion and the formation
of multinucleated
fibers, DMEM supplemented
with 2% horse serum
was used. To reduce the proportion
of unfused myoblasts
further,
cytosine
arabinoside
(10m5 M) was added to the cultures at confluence. Two to three days after the onset of cell fusion, muscle fibers
exhibited spontaneous
contraction.
Human muscle cells were pure
populations
of muscle cells isolated and grown according
to methods
previously
described
(Blau and Webster,
1981).
Nonmuscle
cells were human amniotic fluid cells (amniocytes)
generously
provided by Dr. W. Loughman. Amniocytes
were obtained
from three individuals following amniocentesis
for prenatal diagnosis
and determination
of a normal euploid karyotype.
Amniocytes
consist
of three distinct cell types, AF, F and E, distinguishable
in culture by
their morphology
and by their collagen synthetic
pattern (Hoehn et
al., 1974; Crouch and Bornstein,
1978). Using both these criteria
(data not shown), we identified the fibroblastic
F amniocytes
of three
individuals for use in heterocaryon
experiments.
Prior to fusion with
myotubes.
amniocytes
were grown in the C2 growth medium described above.
Polyethylene
glycol (PEG) was chosen as the fusion agent because
of its simplicity and reproducibility
relative to other agents. Cultures
were treated with PEG 1000 at a concentration
of 50% (w/v)
in
DMEM. pH 7.4, for 60 set at 37”C, according
to the method of
Davidson et al. (1976). Inactivated
Sendai virus was not used as a
fusion agent, in order to eliminate the possibility that the virus might
in some way induce gene expression.
The addition of dimethylsulfoxide to PEG increased
neither the viability of muscle-amniocyte
heterocaryons
nor the frequency
of cell fusion, as it appears to do
with other cell types (Norwood
et al., 1976).
All media were obtained from Grand Island Biologicals.
The horse
serum was from Kansas City Biologicals,
and fetal calf serum from
Sterile Systems,
Logan, Utah. Tissue culture plasticware
was from
Falcon. Cells were grown at 37°C in a humidified Forma incubator
with 10% CO? in air.
Nuclear Identification
Heterocaryons
grown on collagen coated (calf skin coliagen, Calbiochem. 1.4 mg/ml dHnO. autoclaved)
35 mm dishes were washed
three times with Hanks’ balanced salt solution and fixed first in 1%
paraformaldehyde
in Hanks’ solution for 20 min at 37°C and then in
100% methanol for 20 min at -20°C.
Dishes were then rinsed
extensively
with dH*O and stained with Hoechst 33258 (Riedel-de
Haen, Hannover.
Germany),
0.12 Pg/ml 0.9% NaCl for 1.5 min at
37°C. Stained nuclei were visualized in a Leitz fluorescence
microscope with illumination at 340-380
nm.
Extraction
and Identification
of Contractile
Proteins
Contractile
proteins were partially purified from tissues and from cell
cultures by repeated cycles of high salt solubilization
and low salt
precipitation
according
to a modification of the method of Kielley and
Harrington
(1960). Newly synthesized
contractile
proteins were labeled in cultured
cells for 3 hr with 250 pCi/ml ?S-methionine
(Amersham)
prior to extraction,
fractionated
on two-dimensional
gels
(O’Farrell,
1975) and visualized
by autoradiography.
Two-dimensional
polyacrylamide
gel electrophoresis
was performed as described
by O’Farrell (1975), with the exception that the
second-dimension
gel contained
12% acrylamide.
Electrophoretic
transfer of proteins from SDS gels to unmodified
nitrocellulose
was
performed
overnight at 250 mamp. as described
by Burnette (1981).
For immunodetection
of the transferred
proteins, a modified procedure of Burnette
(1981)
was followed.
The first antibody
was a
monoclonal antibody to myosin light chains, designated
T14 (Crow et
al., 1983), generously
provided
by Drs. Crow and Stockdale.
The
second antibody was linked to biotin and reacted with an avidinbiotin horseradish
peroxidase
(Vectastain.
Vector Labs). Antigenantibody complexes
were visualized by reacting the bound horseradish peroxidase
with diaminobenzidine
and H202 according
to the
method of Graham and Karnovsky
(1966).
CK Activity Assay and Gel Electrophoresis
Cell cultures were washed three times with PBS at 4°C and harvested
into glycyl-glycine
buffer (0.05 M glycylglycine.
pH 7.5, 1% NP40,
0.1% BME at 4°C) by scraping with a rubber policeman.
The cell
suspensions
were disrupted
by sonication,
and the supernatants
obtained after centrifugation
were used for analysis of CK activity and
isozyme composition.
Total CK activity was determined
spectrophotometrically
by a
coupled enzyme reaction as previously
described
(Blau and Epstein,
1979) using reagents from Boehringer
Mannheim. One unit of CK is
the amount that catalyzes
the formation of 1 pmole/min
of NADPH at
30°C. All measurements
of CK activity were corrected
for myokinase
(adenylate
kinase) activity
by subtracting
the enzyme activity observed in the absence of the substrate,
creatine phosphate.
Enzyme
activity was expressed
as a function of total protein determined
for
the same samples by the method of Lowry et al. (1951).
CK isozymes were visualized
by a modification
of the method of
Perriard
et al. (1978).
Cell extracts
were electrophoresed
under
nondenaturing
conditions
using a 5% polyacrylamide
slab gel, pH 8.9
(300 V, 6 hr. 4°C). CK isozymes
were detected
in gels with an
agarose overlay (0.5%) impregnated
with the same reaction mixture
Gene Activation
1179
as described
nm).
in Stable
above,
Heterocaryons
and visualized
by ultraviolet
illumination
(366
Acknowledgments
We wish to thank Dr.% Michael Crow and Frank Stockdale
for the use
of their monoclonal
antibody
to the myosin light chains, and Drs.
David Yaffe and William Loughman for providing
us with the mouse
muscle cell line and amniotic fluids cells, respectively.
We are grateful
to Drs. Peter Byers, Laurence Kedes and Frank Stockdale
for critical
reading of the manuscript,
to Drs. Charles Epstein and Douglas
Wallace
for valuable discussions,
and to Cindy Spain and Faith
Lindsay-Fink
for expert secretarial
assistance.
This work was supported by a grant to H. M. B. from the National Institutes of Health
(GM2761 6).
The costs of publication of this article were defrayed in part by the
payment
of page charges.
This article must therefore
be hereby
marked “advertisement”
in accordance
with 18 U.S.C. Section 1734
solely to indicate this fact.
Received
December
14. 1982;
revised
February
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