muscle tumor1 differentiation and control of mitosis in a …...muscle differentiation in a tumor...

[CANCER RESEARCH 30, 596 600, MARCH 1970]

Differentiation and Control of Mitosis in a SkeletalMuscle 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.], andLaboratory of Biology, National Cancer Institute, Bethesda, Maryland 20014 [P. A.}

SUMMARY

A spontaneous tumor in a BALB/cAnN mouse contained two cell types: multinucleated, cross-striated skeletal muscle fibers and mononucleated, nonstriated cells.Mitotic figures were observed only in mononucleatedcells and never in multinucleated units. The tumor wastransplanted subcutaneously or intraperitoneally 5 timesat 2- to 3-month intervals. It continued to form musclefibers and its histolÃ³gica! appearance did not change. Invitro, mononucleated cells liberated from the tumor incorporated thymidine-'H and proliferated. Multinucleated

myotubes arose from the mononucleated cells, and nucleiin these myotubes were diploid and did not incorporatethymidine-'H. Mitoses in vitro were observed only in

mononucleated cells. It is concluded that (a) muscle differentiation in the tumor occurs by the same processeswhich operate in normal myogenesis; (b) nuclei in mono-nucleated tumor cells have lost the ability to respond toenvironmental factors which suppress division in normalmyogenic cells; (c) tumor myotube nuclei derived frommononucleated cells retain the capacity to respond to in-tracellular factors which suppress DNA synthesis andmitosis in normal fibers.

INTRODUCTION

Nuclei in normal multinucleated skeletal muscle fibersdo not synthesize DNA and do not undergo mitosis (4,20, 22). The mechanism of suppression of mitosis in suchnuclei is not known. The presence of myosin in multi-nucleated units and the absence of this protein in dividingmononucleated precursor cells have led to the suggestionthat DNA synthesis and contractile protein synthesis aremutually exclusive processes; i.e., these 2 activities arecoupled in such a way that both cannot simultaneouslyoccur in the same cell (7, 14, 20). Whether or not thishypothesis is correct, it is clear that there is an intrinsicmechanism in multinucleated units for suppressing nuclear replication (see, however, Refs. 5, 11, 23). Normalmononucleated muscle precursor cells are also subject to

1This study was supported in part by Research Contract 3A-6II02B7IR-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.

mitotic suppression (13). In this case, an interplay between the mitotic mechanism of the cell and environmental (extracellular) factors is apparently responsible forthe observed suppression.

With these observations in mind, we were promptedby the discovery of an unusual transplantable skeletalmuscle tumor in a mouse to ask the following questions:(a) In the tumor multinucleated muscle fibers, can nucleisynthesize DNA and divide? (b) Which cells propagatethe tumor and which contain specific muscle proteins?Our observations suggest that nuclei in tumor musclefibers are mitotically suppressed, mononucleated cellsdivide and propagate the tumor, and dividing cells do notsynthesize muscle proteins. In short, myogenesis in thistumor proceeds in the same manner as does the normaldifferentiation of muscle.

MATERIALS AND METHODS

A slowly growing mass appeared spontaneously in theright iliofemoral region of a 6-week-old female BALB/cAnN mouse/ In 8 weeks this mass reached a diameterof approximately 3 cm. The mouse appeared to be unaffected by the tumor except for mechanical difficulties inmovement resulting from the presence of the large massin its leg.

Transplantation of the tumor was carried out by inoculating BALB/cAnN female mice with approximately0.2 ml minced tumor either s.c. or i.p. At the time oftransplantation, tissue was taken for light microscopy,electron microscopy (18), and tissue culture.

Tissue cultures were prepared by incubating smallfragments of tumor in a trypsin-collagenase mixture asdescribed previously (13). The enzyme-treated fragmentswere drawn rapidly through a Pasteur pipet to dispersethe cells, and the resulting suspension was passed througha double layer of lens paper mounted in a Swinny hypodermic adapter (The Millipore Corporation, Bedford,Mass.) to remove multinucleated units and most of theclumps of cells. More than 99% of the cells in the finalsuspension were mononucleated as assayed by examination of stained smears of freshly liberated cells. Mono-nucleated cells were plated onto 22-mm square coverslipscoated 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 thisstudy.

596 CANCER RESEARCH VOL. 30

Research. on February 1, 2021. © 1970 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

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 part200 HIM L-glutamine, 0.1 part Fungizone, 0.1 part penicillin-streptomycin mixture). Culture vessels were 35-mmplastic Petri plates (Falcon Plastics, Los Angeles, Calif.).Plates were incubated at 37.5Â°in water-saturated, 5%CO^-95% air atmosphere. As controls, leg muscles from19-day mouse embryos, from 6-month-old adult mice,and from adult mice 2 to 5 days after the muscles were injured with low temperature (16) were subjected to thesame procedures used for isolation and culture of tumormononucleated cells.

For autoradiography, cultures were incubated for 2 hrin 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 3washes with Earle's balanced salt solution, cultures wereincubated for 30 min in medium containing 0.2 mM unla-beled thymidine. At the end of the chase, cultures wereimmediately fixed or were permitted to incubate for anadditional 16 hr before fixation. Coverslips were mountedon slides with the cells up and were coated with KodakNTB2 liquid emulsion. After 1 to 2 weeks at 4Â°the auto-radiograms were developed in Microdol-X and stainedwith Harris' hematoxylin.

Quantitative estimates of the amount of DNA per nucleus were made on Feulgen-stained cultures. Tumor cultures, mouse embryo muscle cultures, and smears of adultmouse peripheral blood were fixed with alcohol .-formalin:

acetic acid (20:2:1), hydrolyzed in l N HC1 for 15 minat 60Â°,stained in Schiffs reagent for 45 min, and counter-stained with fast green. Coverslips mounted on slideswere examined with a Barr and Stroud GN2 IntegratingMicrodensitometer. Nuclei were observed and measured through a 100X oil immersion objective at a wavelength of 560 TC\H.Three successive readings were takenof each nucleus and of a clear field or part of a cell without a nucleus. Measurements were made on 100 nucleiin tumor myotubes, 100 nuclei in mononucleated cells inthe tumor cultures, 50 nuclei in embryonic muscle myotubes, 50 nuclei in mononucleated cells in embryonicmuscle cultures, and 50 nuclei of mononucleated whiteblood cells in peripheral blood smears.

RESULTS

Light microscopic examination of the original tumor(Fig. 1) revealed that it consisted of 2 types of cells: mul-tinucleated fibers with typical muscle cross-striations andspindle-shaped or round mononucleated cells with no detectable cross-striations. The multinucleated units did notlie in the same plane for their entire length and manywere folded back on themselves. For this reason, it wasnot possible to rule out conclusively that some mononucleated cells were cross-striated. Tangential sectioningof a portion of a multinucleated cell could result in an apparent mononucleated cell with striations. No inflammatory reaction was noted in the tumor and no other his-

tologically recognizable cell types were found. Mincedtissue was transplanted s.c. or i.p. into young female miceof the same strain (BALB/cAnN). Tumors arose in allanimals and were histologically indistinguishable from theoriginal mass. The tumor is now in its 5th passage andhas remained biologically and cytologically similar to theoriginal tumor. Autopsy examination of animals at eachpassage has revealed no mÃ©tastases.

Since tumors may result from an alteration in the control mechanisms which normally prevent DNA synthesisand mitosis, it seemed possible, a priori, that the nucleiin the multinucleated fibers were dividing or synthesizingDNA. Examination of sections revealed that mitotic figures were absent from multinucleated fibers and couldonly be found in nonstriated mononucleated cells. Electron microscopy confirmed the light microscopic observations (18). Multinucleated fibers were observed containing well-organized myofibrils as well as disorganizedarrays of thick (110 to 150 A) and thin (40 to 60 A) filaments. The sarcoplasmic reticulum was present but alsowas not well organized. Nuclei in the multinucleatedunits were often markedly deformed but no mitotic figures were observed in such fibers. These observationssuggested that the controls which operate in normal multinucleated cells to suppress mitosis were also operatingin fibers formed in the tumor. Because of the difficultyin unambiguously identifying a mononucleated cell in asection, however, cell cultures were initiated to studyDNA synthesis and mitosis in an essentially 2-dimen-sional system.

Mononucleated cells from a 4th passage tumor wereplated onto collagen-coated coverslips. Cells attached tothe substrate and began to multiply by the 2nd day invitro. Four to 6 days after plating, multinucleated myotubes started to form. Mitotic figures were observed atthis time only in mononucleated cells. In examininghundreds of nuclei which were unequivocally in myotubes,no instance of a mitotic figure was found. Cultures whichwere 1 to 5 days old were exposed to thymidine-3H for

2 hr. Examination of autoradiograms of cultures fixed immediately after exposure to the isotope (Figs. 2 and 3)showed incorporation of label into nuclei of mononucleated cells. No label was detected in nuclei which wereinside myotubes. Occasionally, mononucleated cells withlabeled nuclei were observed on top of multinucleatedunits (Fig. 4). In these cases, the outlines of the mono-nucleated cells could usually be discerned. In cultureswhich had been exposed to thymidine-3H for 2 hr and

were permitted to incubate for an additional 16 hr afterfixation, both labeled and unlabeled nuclei were observedin myotubes as well as in mononucleated cells. Continued growth resulted in increased length of myotubesand further proliferation of mononucleated cells. Rarely,contractions were observed in multinucleated fibers. Cultures initiated with mononucleated cells from normaladult muscle or regenerating adult muscle formed lessthan 1% of the number of myotubes in tumor cultures.

Cultures at Days 4 to 6 were fixed and stained by theFeulgen reaction. Quantitative cytophotometric meas-

MARCH 1970 597



Nameroff, Reznik, Anderson, and Hansen

40

o

oQÂ£

20

10

MYOTUBES

MONONUCLEATEDCELLS

10 20 30 50

2N 4N

AMOUNT OF DNA (absorption units)

Chart I. Cultures containing mononucleated cells and myotubeswere stained by the Feulgen reaction. Quantitative estimates of theamount of DNA in individual nuclei in myotubes and mononucleatedcells are depicted. O -O. mononucleated cells; 9 9, nuclei inmyotubes. 2N, diploid amount of DNA determined from peripheralblood leukocytes or normal myotube nuclei. 4N, value for metaphasefigures in normal mononucleated cells in vitro.

urements were made on individual nuclei in both mono-nucleated cells and myotubes (Chart 1). DNA values fornuclei in myotubes were grouped around the diploid(2N) amount, with peripheral blood mononuclear cells ornuclei in normal embryo myotubes in vitro as references.The few values higher than the 2N amount can be attributed to abnormal (perhaps heteroploid) nuclei inmyotubes or, more likely, to nuclei in mononucleatedcells which were synthesizing DNA and were lying on orunder myotubes (Fig. 4). DNA values for nuclei in mono-nucleated cells ranged from the 2N to the 4N amountas would be expected in a population of dividing cells.

DISCUSSION

The data presented here suggest that muscle differentiation in the tumor proceeds by the same mechanismsthat operate in normal myogenesis [see review by KÃ¶nigsberg (9)]. DNA synthesis and multiplication of myotubenuclei by mitosis can be ruled out, since myotube nucleiare diploid and do not incorporate thymidine-3H. Fusion

of mononucleated cells therefore appears to be the mechanism responsible for multinucleation and is supportedby the finding of labeled nuclei in myotubes 16 hr aftera brief exposure of cultures to thymidine-'H. Electron

microscopic examination (18) suggests that, as in normalmyogenesis, contractile proteins are synthesized by non-dividing tumor cells (7, 17). Myofibrillar filaments werenot observed in cells containing mitotic figures.

It is apparent, then, that the mononucleated cells arepropagating the tumor, since nuclei in multinucleated

units have withdrawn from the mitotic cycle. This behavior is different from that of normal mononucleatedmuscle precursor cells which do not indefinitely propagateduring the formation of a muscle. Normal myogenic cellsare subject to extracellular influences which result in cessation of mitosis (13). It appears, therefore, that the tumormononucleated cells have an altered ability to respond toenvironmental factors which discourage cell division. Thenature of the metabolic alteration which led to transformation to the tumor state in mononucleated cells is unknown.It remains unclear why a nucleus in a mononucleatedtumor cell does not respond to the normal suppressorsof mitosis while a nucleus with the same genetic information does respond when it is in a multinucleated myotube.It is conceivable that, during the cell division in whichthe decision to fuse is made [the so-called "quantal mitosis" (8)], the presumed metabolic derangement is lost or

becomes inoperative. According to this notion, cells whichcan fuse are no longer abnormal and the muscle fibersin the tumor are not metabolically deranged, but theirlack of attachment and unusual location result in the apparent morphological abnormalities. Whatever the defectin these cells may be, however, it is clear that the controlof mitosis in mononucleated precursor cells can be separated from the control operating in multinucleated units;i.e., the former can be lost or altered without affecting thelatter.

Tumors containing muscle fibers have been describedin the literature in both human patients and experimentalanimals (1-3, 10, 12). Several such tumors (1, 12) contained other cell types such as cartilage or areas of myx-omatous tissue along with the muscle. Previous studiesof "rhabdomyosarcomas" both in vivo and in vitro (2, 21)

are subject to the criticism that fibers found in such tumors may have arisen by the process of regeneration frompreexisting mononucleated cells which were not themselves part of the tumor. In most of the reported studiesit has not been demonstrated that transformed tumorcells produced new muscle fibers. In the tumor describedin this paper, new muscle continues to arise in transplanted tumors initiated from small fragments of tissueand muscle fibers continue to be the predominant celltype found. Hence, it is unlikely that the fibers in thetumor arose from normal mononucleated cells carriedover from the original tumor.

In the present system, as in other tumor systems reported in the literature (19), altered precursor cells propagate the tumor while the histologically recognizablecell types (the terminally differentiated cells) do not appear to be capable of indefinite proliferation. Thus, it maybe a general phenomenon that tumors increase in cellnumber by multiplication of the cells which do not formrecognizable tissue types in the mass. Although patholo-gists name a tumor, in part, by the tissue type which theyobserved in it, it is very difficult to assign a particularphenotype 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, whenpresent in a tumor, may be altered in their metabolism




Muscle Differentiation in a Tumor

(15) but may have nothing to do with tumor proliferation.

REFERENCES

1. Bullock, F. D., and Curtis, M. R. A Transplantable MetastasizingChondro-Rhabdo-Myo-Sarcoma of the Rat. J. Cancer Res., 7:195-207, 1922.

2. Corbeil, L. B. Differentiation of Rhabdomyosarcoma and NeonatalMuscle Cells in Vilro. Cancer, 20: 572-578, 1967.

3. Cornog, J. L., and Gonatas, N. K. Ultrastructure of a Rhabdomy-oma. J. Ultrastruct. Res., 20: 433-450, 1967.

4. Firket, H. Recherches sur la SynthÃ¨sedes Acides DÃ©soxyribonu-clÃ©iqueset la PrÃ©parationÃ la Mitose dans des Cellules CultivÃ©esÂ¡nVitro (Etude CytophotomÃ©triqueet Autoradiographique).Arch. Biol. (LiÃ¨ge),69: 1 166, 1958.

5. Fogel, M., and Defendi, V. Infection of Muscle Cultures from Various Species with Oncogenic DNA Viruses (SV40 and Polyoma).Proc. Nati. Acad. Sei. U. S., 58: 967-973, 1967.

6. Hauschka, S., and KÃ¶nigsberg, I. R. The Influence of Collagenon the Development of Muscle Clones. Proc. Nati. Acad. Sei. U. S.,55: 119-126, 1966.

7. Holtzer, H., Marshall, J. M., and Finck, H. An Analysis of Myo-genesis by the Use of Fluorescent Antimyosin. J. Biophys. Bio-chem. Cytol., 3: 705-724, 1957.

8. Ishikawa, H.. Bischoff, R., and Holtzer, H. Mitosis and Intermediate-sized Filaments in Developing Skeletal Muscle. J. Cell Biol.,38: 538-555, 1968.

9. KÃ¶nigsberg,I. R. Aspects of Cytodifferentiation of Skeletal Muscle.In: R. L. DeHaan and H. Ursprung (eds.), OrganogÃ©nesis,pp.337 358. New York: Holt, Rinehart, and Winston, 1965.

10. Kroll, A., Kuwabara, T., and Howard, G. Electron Microscopy ofRhabdomyosarcoma of the Orbit. Invest. Ophthalmol., 2: 523-537,

1963.11. Lee, H. H., Kaighn, M. E., and Ebert, J. D. Induction of Thymi

dine-'H Incorporation in Multinucleated Myotubes by Rous Sarcoma Virus. Intern. J. Cancer, 3: 126-136, 1968.

12. Maddock, C. L., Kury, L., and Riley, E. A Transplantable Metastasizing Rhabdomyosarcoma (Mesenchymoma) in the W/Fu Rat.Cancer Res., 22: 291-293, 1962.

13. NamerofT, M., and Holtzer, H. Contact-mediated Reversible Suppression of Myogenesis. Develop. Biol., 19: 380 396, 1969.

14. Okazaki. K., and Holtzer, H. An Analysis of Myogenesis in VitroUsing Fluorescein-labeled Antimyosin. J. Histochem. Cytochem..13: 726-739, 1965.

15. Potter, V. R., Watanabe, M., Pilot, H. C., and Morris, H. P. Systematic Oscillations in Metabolic Activity in Rat Liver and Hepa-tomas. Survey of Normal Diploid and Other Hepatomas Lines.Cancer Res., 29: 55-78. 1969.

16. Price, H. M., Howes, E. L., and Blumberg, J. M. Ultrastructural Alterations in Skeletal Muscle Fibers Injured by Cold. I. The AcuteDegenerative Changes. Lab. Invest., 13: 1264-1278, 1964.

17. Przybylski, R. J., and Blumberg, J. M. Ultrastructural Aspects ofMyogenesis in the Chick. Lab. Invest., 15: 836-863, 1966.

18. Reznik, M., Nameroff, M., and Hansen, J. L. Ultrastructure of aTransplantable Murine Rhabdomyosarcoma. Cancer Res., 30:601-610, 1970.

19. Stevens, L. C. The Biology of Teratomas. Advan. Morphogenesis,6: 1-31, 1967.

20. Stockdale, F. E., and Holtzer, H. DNA Synthesis and Myogenesis.Exptl. Cell Res., 24: 508 520, 1961.

21. Stout, A. P.. and Lattes, R. Malignant Tumors of Muscle. In: A. P.Stout (ed.), Tumors of the Soft Tissues, pp. 134-144. Washington,D. C.: Armed Forces Institute of Pathology, 1967.

22. Strehler, B. L., KÃ¶nigsberg,I. R., and Kelley, J. E. T. Ploidy ofMyotube Nuclei Developing in Vitro as Determined with a Recording Double Beam Microspectrophotometer. Exptl. Cell Res...52:232-241, 1963.

23. Yaffe, D., and Gershon, D. Multinucleated Muscle Fibers: Induction of DNA Synthesis and Mitosis by Polyoma Virus Infection.Nature, 2/5:421-424, 1967.

Fig. 1. Typical field in a section of tumor. Cross-striations are visible in many cells. Nuclei appear to be centrally located in multinucleatedfibers. Myofibrils are largely in register but are not straight. This appearance is probably a result of the lack of proper end attachment and concomitant 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. Theirnuclei 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 acell over a myotube. Several unlabeled myotube nuclei are indicated by Arrows B. X 220.

MARCH 1970 599



Nameroff *Reznik* Anderson, and Hansen

Â«*




1970;30:596-600. Cancer Res Mark A. Nameroff, Michel Reznik, Paul Anderson, et al. TumorDifferentiation and Control of Mitosis in a Skeletal Muscle

Updated version

http://cancerres.aacrjournals.org/content/30/3/596

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/30/3/596To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



muscle tumor1 differentiation and control of mitosis in a …...muscle differentiation in a tumor...

Documents