[CANCER RESEARCH 30, 596 600, MARCH 1970]
Differentiation and Control of Mitosis in a Skeletal
Muscle Tumor1
Mark A. Nameroff, Michel Reznik,
Paul Anderson, and James L. Hansen
Laboratory of Skeletal Muscle Research. Armed Forces Institute of Pathology, Washington, D. C. 20305 [M. A. N., M. R.,J. L. H.], and
Laboratory of Biology, National Cancer Institute, Bethesda, Maryland 20014 [P. A.}
SUMMARY
A spontaneous tumor in a BALB/cAnN mouse con
tained two cell types: multinucleated, cross-striated skele
tal muscle fibers and mononucleated, nonstriated cells.
Mitotic figures were observed only in mononucleated
cells and never in multinucleated units. The tumor was
transplanted subcutaneously or intraperitoneally 5 times
at 2- to 3-month intervals. It continued to form muscle
fibers and its histológica! appearance did not change. In
vitro, mononucleated cells liberated from the tumor in
corporated thymidine-'H and proliferated. Multinucleated
myotubes arose from the mononucleated cells, and nuclei
in these myotubes were diploid and did not incorporate
thymidine-'H. Mitoses in vitro were observed only in
mononucleated cells. It is concluded that (a) muscle dif
ferentiation in the tumor occurs by the same processes
which operate in normal myogenesis; (b) nuclei in mononucleated tumor cells have lost the ability to respond to
environmental factors which suppress division in normal
myogenic cells; (c) tumor myotube nuclei derived from
mononucleated cells retain the capacity to respond to intracellular factors which suppress DNA synthesis and
mitosis in normal fibers.
INTRODUCTION
Nuclei in normal multinucleated skeletal muscle fibers
do not synthesize DNA and do not undergo mitosis (4,
20, 22). The mechanism of suppression of mitosis in such
nuclei is not known. The presence of myosin in multinucleated units and the absence of this protein in dividing
mononucleated precursor cells have led to the suggestion
that DNA synthesis and contractile protein synthesis are
mutually exclusive processes; i.e., these 2 activities are
coupled in such a way that both cannot simultaneously
occur in the same cell (7, 14, 20). Whether or not this
hypothesis is correct, it is clear that there is an intrinsic
mechanism in multinucleated units for suppressing nu
clear replication (see, however, Refs. 5, 11, 23). Normal
mononucleated muscle precursor cells are also subject to
1This study was supported in part by Research Contract 3A6II02B7IR-02 from the Medical Research and Development Command,
U. S. Army, Washington, D. C.
"'Recipient of USPHS International Postdoctoral Research Fellow
ship F05-TW-1169.02.
Received May 5, 1969; accepted July 18, 1969.
596
mitotic suppression (13). In this case, an interplay be
tween the mitotic mechanism of the cell and environ
mental (extracellular) factors is apparently responsible for
the observed suppression.
With these observations in mind, we were prompted
by the discovery of an unusual transplantable skeletal
muscle tumor in a mouse to ask the following questions:
(a) In the tumor multinucleated muscle fibers, can nuclei
synthesize DNA and divide? (b) Which cells propagate
the tumor and which contain specific muscle proteins?
Our observations suggest that nuclei in tumor muscle
fibers are mitotically suppressed, mononucleated cells
divide and propagate the tumor, and dividing cells do not
synthesize muscle proteins. In short, myogenesis in this
tumor proceeds in the same manner as does the normal
differentiation of muscle.
MATERIALS
AND METHODS
A slowly growing mass appeared spontaneously in the
right iliofemoral region of a 6-week-old female BALB/
cAnN mouse/ In 8 weeks this mass reached a diameter
of approximately 3 cm. The mouse appeared to be unaf
fected by the tumor except for mechanical difficulties in
movement resulting from the presence of the large mass
in its leg.
Transplantation of the tumor was carried out by inocu
lating BALB/cAnN female mice with approximately
0.2 ml minced tumor either s.c. or i.p. At the time of
transplantation, tissue was taken for light microscopy,
electron microscopy (18), and tissue culture.
Tissue cultures were prepared by incubating small
fragments of tumor in a trypsin-collagenase mixture as
described previously (13). The enzyme-treated fragments
were drawn rapidly through a Pasteur pipet to disperse
the cells, and the resulting suspension was passed through
a double layer of lens paper mounted in a Swinny hypo
dermic adapter (The Millipore Corporation, Bedford,
Mass.) to remove multinucleated units and most of the
clumps of cells. More than 99% of the cells in the final
suspension were mononucleated as assayed by examina
tion of stained smears of freshly liberated cells. Mononucleated cells were plated onto 22-mm square coverslips
coated with collagen (6) at a concentration of 0.3 X IO6
1The "Principles of Laboratory Animal Care" as promulgated by
the National Society for Medical Research were observed during this
study.
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Muscle Differentiation in a Tumor
cells in 1.5 ml tissue culture medium (7.7 parts Eagle's
minimal essential medium, 2 parts horse serum, 0.1 part
200 HIM L-glutamine, 0.1 part Fungizone, 0.1 part peni
cillin-streptomycin mixture). Culture vessels were 35-mm
plastic Petri plates (Falcon Plastics, Los Angeles, Calif.).
Plates were incubated at 37.5°in water-saturated, 5%
CO^-95% air atmosphere. As controls, leg muscles from
19-day mouse embryos, from 6-month-old adult mice,
and from adult mice 2 to 5 days after the muscles were in
jured with low temperature (16) were subjected to the
same procedures used for isolation and culture of tumor
mononucleated cells.
For autoradiography, cultures were incubated for 2 hr
in medium containing thymidine-'H (specific activity,
3.0 Ci/mmole, Schwarz BioResearch, Inc., Orangeburg,
N. Y.) at a concentration of 0.5 ^Ci/ml. Following 3
washes with Earle's balanced salt solution, cultures were
incubated for 30 min in medium containing 0.2 mM unlabeled thymidine. At the end of the chase, cultures were
immediately fixed or were permitted to incubate for an
additional 16 hr before fixation. Coverslips were mounted
on slides with the cells up and were coated with Kodak
NTB2 liquid emulsion. After 1 to 2 weeks at 4°the autoradiograms were developed in Microdol-X and stained
with Harris' hematoxylin.
Quantitative estimates of the amount of DNA per nu
cleus were made on Feulgen-stained cultures. Tumor cul
tures, mouse embryo muscle cultures, and smears of adult
mouse peripheral blood were fixed with alcohol .-formalin:
acetic acid (20:2:1), hydrolyzed in l N HC1 for 15 min
at 60°,stained in Schiffs reagent for 45 min, and counterstained with fast green. Coverslips mounted on slides
were examined with a Barr and Stroud GN2 Integrating
Microdensitometer.
Nuclei were observed and meas
ured through a 100X oil immersion objective at a wave
length of 560 TC\H.Three successive readings were taken
of each nucleus and of a clear field or part of a cell with
out a nucleus. Measurements were made on 100 nuclei
in tumor myotubes, 100 nuclei in mononucleated cells in
the tumor cultures, 50 nuclei in embryonic muscle myo
tubes, 50 nuclei in mononucleated cells in embryonic
muscle cultures, and 50 nuclei of mononucleated white
blood cells in peripheral blood smears.
RESULTS
Light microscopic examination of the original tumor
(Fig. 1) revealed that it consisted of 2 types of cells: multinucleated fibers with typical muscle cross-striations and
spindle-shaped or round mononucleated cells with no de
tectable cross-striations. The multinucleated units did not
lie in the same plane for their entire length and many
were folded back on themselves. For this reason, it was
not possible to rule out conclusively that some mononu
cleated cells were cross-striated. Tangential sectioning
of a portion of a multinucleated cell could result in an ap
parent mononucleated cell with striations. No inflamma
tory reaction was noted in the tumor and no other hisMARCH
tologically recognizable cell types were found. Minced
tissue was transplanted s.c. or i.p. into young female mice
of the same strain (BALB/cAnN). Tumors arose in all
animals and were histologically indistinguishable from the
original mass. The tumor is now in its 5th passage and
has remained biologically and cytologically similar to the
original tumor. Autopsy examination of animals at each
passage has revealed no métastases.
Since tumors may result from an alteration in the con
trol mechanisms which normally prevent DNA synthesis
and mitosis, it seemed possible, a priori, that the nuclei
in the multinucleated fibers were dividing or synthesizing
DNA. Examination of sections revealed that mitotic fig
ures were absent from multinucleated fibers and could
only be found in nonstriated mononucleated cells. Elec
tron microscopy confirmed the light microscopic observa
tions (18). Multinucleated fibers were observed contain
ing well-organized myofibrils as well as disorganized
arrays of thick (110 to 150 A) and thin (40 to 60 A) fila
ments. The sarcoplasmic reticulum was present but also
was not well organized. Nuclei in the multinucleated
units were often markedly deformed but no mitotic fig
ures were observed in such fibers. These observations
suggested that the controls which operate in normal mul
tinucleated cells to suppress mitosis were also operating
in fibers formed in the tumor. Because of the difficulty
in unambiguously identifying a mononucleated cell in a
section, however, cell cultures were initiated to study
DNA synthesis and mitosis in an essentially 2-dimensional system.
Mononucleated cells from a 4th passage tumor were
plated onto collagen-coated coverslips. Cells attached to
the substrate and began to multiply by the 2nd day in
vitro. Four to 6 days after plating, multinucleated myo
tubes started to form. Mitotic figures were observed at
this time only in mononucleated cells. In examining
hundreds of nuclei which were unequivocally in myotubes,
no instance of a mitotic figure was found. Cultures which
were 1 to 5 days old were exposed to thymidine-3H for
2 hr. Examination of autoradiograms of cultures fixed im
mediately after exposure to the isotope (Figs. 2 and 3)
showed incorporation of label into nuclei of mononu
cleated cells. No label was detected in nuclei which were
inside myotubes. Occasionally, mononucleated cells with
labeled nuclei were observed on top of multinucleated
units (Fig. 4). In these cases, the outlines of the mononucleated cells could usually be discerned. In cultures
which had been exposed to thymidine-3H for 2 hr and
were permitted to incubate for an additional 16 hr after
fixation, both labeled and unlabeled nuclei were observed
in myotubes as well as in mononucleated cells. Con
tinued growth resulted in increased length of myotubes
and further proliferation of mononucleated cells. Rarely,
contractions were observed in multinucleated fibers. Cul
tures initiated with mononucleated cells from normal
adult muscle or regenerating adult muscle formed less
than 1% of the number of myotubes in tumor cultures.
Cultures at Days 4 to 6 were fixed and stained by the
Feulgen reaction. Quantitative cytophotometric meas-
1970
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597
Nameroff, Reznik, Anderson, and Hansen
40
MYOTUBES
o
o
Q£
20
MONONUCLEATED
CELLS
10
10
20
30
50
2N
AMOUNT OF DNA (absorption
4N
units)
Chart I. Cultures containing mononucleated cells and myotubes
were stained by the Feulgen reaction. Quantitative estimates of the
amount of DNA in individual nuclei in myotubes and mononucleated
cells are depicted. O
-O. mononucleated cells; 9
9, nuclei in
myotubes. 2N, diploid amount of DNA determined from peripheral
blood leukocytes or normal myotube nuclei. 4N, value for metaphase
figures in normal mononucleated cells in vitro.
urements were made on individual nuclei in both mononucleated cells and myotubes (Chart 1). DNA values for
nuclei in myotubes were grouped around the diploid
(2N) amount, with peripheral blood mononuclear cells or
nuclei in normal embryo myotubes in vitro as references.
The few values higher than the 2N amount can be at
tributed to abnormal (perhaps heteroploid) nuclei in
myotubes or, more likely, to nuclei in mononucleated
cells which were synthesizing DNA and were lying on or
under myotubes (Fig. 4). DNA values for nuclei in mononucleated cells ranged from the 2N to the 4N amount
as would be expected in a population of dividing cells.
DISCUSSION
The data presented here suggest that muscle differ
entiation in the tumor proceeds by the same mechanisms
that operate in normal myogenesis [see review by Königs
berg (9)]. DNA synthesis and multiplication of myotube
nuclei by mitosis can be ruled out, since myotube nuclei
are diploid and do not incorporate thymidine-3H. Fusion
of mononucleated cells therefore appears to be the mech
anism responsible for multinucleation and is supported
by the finding of labeled nuclei in myotubes 16 hr after
a brief exposure of cultures to thymidine-'H. Electron
microscopic examination (18) suggests that, as in normal
myogenesis, contractile proteins are synthesized by nondividing tumor cells (7, 17). Myofibrillar filaments were
not observed in cells containing mitotic figures.
It is apparent, then, that the mononucleated cells are
propagating the tumor, since nuclei in multinucleated
598
units have withdrawn from the mitotic cycle. This be
havior is different from that of normal mononucleated
muscle precursor cells which do not indefinitely propagate
during the formation of a muscle. Normal myogenic cells
are subject to extracellular influences which result in ces
sation of mitosis (13). It appears, therefore, that the tumor
mononucleated cells have an altered ability to respond to
environmental factors which discourage cell division. The
nature of the metabolic alteration which led to transforma
tion to the tumor state in mononucleated cells is unknown.
It remains unclear why a nucleus in a mononucleated
tumor cell does not respond to the normal suppressors
of mitosis while a nucleus with the same genetic informa
tion does respond when it is in a multinucleated myotube.
It is conceivable that, during the cell division in which
the decision to fuse is made [the so-called "quantal mito
sis" (8)], the presumed metabolic derangement is lost or
becomes inoperative. According to this notion, cells which
can fuse are no longer abnormal and the muscle fibers
in the tumor are not metabolically deranged, but their
lack of attachment and unusual location result in the ap
parent morphological abnormalities. Whatever the defect
in these cells may be, however, it is clear that the control
of mitosis in mononucleated precursor cells can be sepa
rated from the control operating in multinucleated units;
i.e., the former can be lost or altered without affecting the
latter.
Tumors containing muscle fibers have been described
in the literature in both human patients and experimental
animals (1-3, 10, 12). Several such tumors (1, 12) con
tained other cell types such as cartilage or areas of myxomatous tissue along with the muscle. Previous studies
of "rhabdomyosarcomas" both in vivo and in vitro (2, 21)
are subject to the criticism that fibers found in such tu
mors may have arisen by the process of regeneration from
preexisting mononucleated cells which were not them
selves part of the tumor. In most of the reported studies
it has not been demonstrated that transformed tumor
cells produced new muscle fibers. In the tumor described
in this paper, new muscle continues to arise in trans
planted tumors initiated from small fragments of tissue
and muscle fibers continue to be the predominant cell
type found. Hence, it is unlikely that the fibers in the
tumor arose from normal mononucleated cells carried
over from the original tumor.
In the present system, as in other tumor systems re
ported in the literature (19), altered precursor cells prop
agate the tumor while the histologically recognizable
cell types (the terminally differentiated cells) do not ap
pear to be capable of indefinite proliferation. Thus, it may
be a general phenomenon that tumors increase in cell
number by multiplication of the cells which do not form
recognizable tissue types in the mass. Although pathologists name a tumor, in part, by the tissue type which they
observed in it, it is very difficult to assign a particular
phenotype to a cell in mitosis in a tissue section. Hence,
there may be no such thing as a malignant chondrocyte,
red blood cell, or hepatocyte, etc. These cell types, when
present in a tumor, may be altered in their metabolism
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Muscle Differentiation in a Tumor
(15) but may have nothing to do with tumor prolifera
tion.
12.
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Fig. 1. Typical field in a section of tumor. Cross-striations are visible in many cells. Nuclei appear to be centrally located in multinucleated
fibers. Myofibrils are largely in register but are not straight. This appearance is probably a result of the lack of proper end attachment and con
comitant absence of tension in fibers. Phosphotungstic acid-hematoxylin, X 220.
Fig. 2. Autoradiogram of a 3-day culture after a 2-hr exposure to thymidine-'H. Only mononucleated cells are present in the culture. About
40% of the cells have incorporated the label, x 220.
Fig. 3. Autoradiogram of a 5-day culture after a 2-hr exposure to thymidine-'H. Myotubes have begun to form. Arrows, nuclei in a myotube.
No label is present in any nuclei which are unequivocally inside a myotube. Many labeled mononucleated cells are present in this field. Their
nuclei appear black. X 145.
Fig. 4. Labeled nuclei were occasionally observed in mononucleated cells lying on top of myotubes. Arrow A points to a labeled nucleus in a
cell over a myotube. Several unlabeled myotube nuclei are indicated by Arrows B. X 220.
MARCH 1970
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Nameroff * Reznik* Anderson, and Hansen
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CANCER RESEARCH
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Differentiation and Control of Mitosis in a Skeletal Muscle
Tumor
Mark A. Nameroff, Michel Reznik, Paul Anderson, et al.
Cancer Res 1970;30:596-600.
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