Transformation is an alternative to normal skeletal muscle development

Printed in Sweden Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/80/020333-17%o2.oo/O

Experimental

TRANSFORMATION

J. KAUFMAN, Department

12.5 (1980) 333-349

IS AN ALTERNATIVE

SKELETAL STEPHEN

Cell Research

MUSCLE

CHRISTINE

TO NORMAL

DEVELOPMENT

M. PARKS, JOSEPH BOHN and LIA E. FAIMAN

of Microbiology and School University of Illinois, Urbana,

of Basic Medical IL 61801, USA

Sciences,

SUMMARY The differentiation of skeletal muscle is characterized by cessation of proliferation and fusion of single myoblasts to form non-replicating multinucleate fibers (myotubes). If termination of proliferation is an obligate requirement for further differentiation, myoblasts defective in this stage of development should fail to fuse or exhibit any further characteristics of myotubes. Furthermore, myoblasts which have lost the ability to control and cease proliferation may represent a transformed, potentially tumorigenic population. Formation of the neoplastic state may therefore be viewed as an alternate path, antithetical to the normal differentiation of skeletal muscle. To test this hypothesis, we isolated 13 clones of non-fusing cells from the myogenic L, line of rat myoblasts. In contrast to the Ls line, all of the non-fusing clones maintain their proliferative capacity, do not form myotubes, nor elevated creatine kinase activity nor increased myosin, but do develop into tumors when injected into athymic mice. LB cells do not produce tumors in these mice. Analysis of cell growth and serum requirements, plasminogen activator, hexose transport, adhesiveness, LETS protein and growth in soft agar, indicates that these non-fusing cells are transformed and clearly distinguished from the parent L, cells. Whereas the L8 line maintains a near diploid complement of chromosomes, ail non-fusing clones were DOlVDlOid. In addition. 12 of 13 non-fusing clones (but not the L, cells) express an endogenous type ‘C v&s. Although all clones defective in differentiation formed tumors, no single in vitro characteristic was found to be a constant index of this tumorigenic capacity. We conclude that cessation of proliferation is an obligate requirement for skeletal myogenesis, that transformation is an alternative to normal skeletal muscle development and that the phenotype of these transformed cells may be quite varied.

The differentiation of skeletal muscle is characterized by the fusion of single myoblasts to form non-replicating multinucleate fibers. Commensurate but not necessarily dependent on fusion is the expression of numerous characteristics of the differentiated state including the synthesis of the structural components of the contractile apparatus (e.g., myosin, a-actin, tropomyosin and troponin) and the muscle-specific enzymes creatine kinase, aldolase and glycogen phosphorylase [for recent reviews see l-31. Acetylcholine receptors and acetylcholinesterase activity also appear in the

fused cells as a prelude to neuronal innervation. These receptors may also be present on single myoblasts [4]. The expression of the differentiated phenotype is generally thought to be temporally controlled and coordinate with the transcription and translation of discrete new species of mRNA [581; however, some evidence suggests that specific mRNA molecules may be synthesized and stored, but not expressed, prior to fusion [9, lo]. Holtzer and coworkers proposed that myogenic cells shift from a replicating to a non-replicating population prior to fusion Exp Cell Res 125 (1980)

334

Kaufman

et al.

(or the expression of other characteristics of the differentiated phenotype) and that this transition is a consequence of biochemical differentiation occurring during the terminal round of myoblast replication [ 11, 121. Alternatively, Konigsberg and colleagues favor a stochastic model for myogenie differentiation in which myoblasts fuse in a probabilistic fashion based upon the length of their Gl period; myoblasts would thus cease replication as a consequence of fusion [13]. In either case termination of the proliferative phase of myogenesis appears to be under stringent control. Although myotubes retain the capacity for DNA repair synthesis [14], their capacity for semiconservative replication is markedly reduced as evidenced by diminished levels of DNA polymerase [15, 161. Myoblasts fuse to form myotubes during the Gl phase of the cell cycle [13, 17, 181, and under normal circumstances, DNA is not synthesized in myotube nuclei [ 19, 201. Thus, the normal development of myoblasts into myotubes represents a course which is incompatible with the unrestricted proliferative capacity that is characteristic of neoplastic growth. Conversely, if termination of proliferation is an obligate requirement for myoblast differentiation [ll, 12, 191, myoblasts defective in this stage of development should fail to fuse or exhibit any further characteristics of myotubes. Furthermore, myoblasts which have lost the ability to control and cease proliferation may represent a transformed, potentially tumorigenic population [2 11. Formation of the neoplastic state may therefore be viewed as an alternate path, antithetical to the normal differentiation of skeletal muscle. To test this hypothesis, we isolated a non-fusing variant, fu-1 , from the L, line of myogenic rat myoblasts. Fu-1 cells do not exhibit properties of differentiated muscle Exp Cdl

Res 125 (1980)

and are distinct from the myogenic L, cells by numerous criteria often associated with transformed growth in vitro [22]. Fu-1 cells, but not L, cells, also form tumors when injected into athymic mice and fu-1 cells express an endogenous type C virus that is not made by L8 cells. To extend these findings and further determine whether the loss of the capacity of myoblasts to differentiate is accompanied by a loss of normal growth control, we selected 12 additional clones of non-fusing cells by several different procedures. We have determined the developmental status of these non-fusing lines as well as their capacity to grow in soft agar, their relative adhesiveness, their rates of hexose transport, the presence of elevated proteolytic activity and quantitative changes in a high molecular weight glycoprotein (LETS or fibronectin) on their plasma membrane. These criteria were chosen as they have often been reported to be properties of chemically and virally transformed cells. In addition, we have determined the tumorigenicity of these cells in vivo and the expression of endogenous virus. Each of the non-fusing cell lines failed to differentiate and exhibited one or more properties of transformed cells in accord with the hypothesis that the loss of proliferative control is tantamount to the loss of the capacity to differentiate.

MATERIALS

AND

METHODS

Cell culture conditions L, cells provided by Yaffe were cloned and maintained by serial passage as previously reported [22]. A highly myogenic clone designated E63 was isolated from the LB line and used in some of these experiments. All cells were grown in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10 % horse serum (ISI). oenicillin (100 U/ml). strentomvcin (100 pg/ml) and’kanamycin (1 pg/ml), at 3Pd and-in a iO% CO, high humidity atmosphere. 0.5% chick embryo extract was initially included in the medium; however, as it is not required for the differentiation of these

Trunsformation cells, it was subsequently omitted. Non-fusing variants were isolated as indicated below. All cells were grown in Falcon tissue culture dishes.

Cell growth and myotube formation Ceils were either counted in a model ZBI Coulter counter or nuclei were enumerated in a hemocytometer after cell lysis in 0.75% acetic acid. To assay myoblast fusion, cells were washed in Dulbecco’s medium, fixed for 20 min in 70% methanol, stained in fresh 4% Giemsa (Harleco) and then rinsed with distilled water. The number of nuclei within fibers was scored in at least 10 fields/plate using an ocular grid.

Isolation

of cell lines

(i) The non-fusing fu-1 line was isolated bv reeeated serial passage (sp) of myoblasts remaining in-highly fused cultures f221: fu-2 and fu-3 were also isolated by this procedure. ?X lo5 cells were plated/50 mm dish; 7 days later unfused myoblasts were collected by trypsinization (0.05% trypsin in Ca2+, Mg*+-free Earle’s salt solution) and replated at the same density. This selection was repeated 5-6 times; the cells were then cloned in microtiter dishes. Clones were passaged twice and assayed for fusion; non-fusing clones were grown and frozen at -76°C at a concentration of IX lo6 cells/ml, in medium containing 20% horse serum and 10 % dimethylsulfoxide and at a cooling rate of approx. l”C/min. (ii) Agar plates (ap); Cells were grown for 4-5 days in 100 mm dishes coated with IO ml I % Difco Bacto agar in Ca*+, Mg2+-free Earle’s solution. These cells grew as grape-like clusters in the medium and were collected by centrifugation, cloned and assayed for fusion. Lines designated fu-5,7 and 9 were then grown and stored as indicated above. (iii) Cells were selected by growth on plastic (p) not pretreated for optimal growth of cells in tissue culture. Growth on this matrix may select for cells with reduced anchorage-dependent growth [23]. These cells were reulated on tissue culture dishes: clones fu-4 and 6 were subsequently isolated. (iv) Spinner culture (sc); 4X10’ L, cells in 20 ml medium were incubated as a gently stirred suspension for 3 days at which time onlv lX105 cells survived. These cells were then grown on tissue culture dishes and clone fu-8 was isolated. (v) Concanavalin A (ConA): Cells were selected based upon their surviving treatment with ConA (Pharmacia). 1.5~ lo5 L, cells grown 24 h on 50 mm dishes were treated for 30 min with I mg/ml ConA in Dulbecco’s phosphate buffered saline, pH 7.3 (PBS). Surviving cells were grown, replated and treated again with I mg/ml ConA. This procedure was repeated and resulted in two populations: cells floating in the medium were centrifuged, plated, treated again with ConA, replated and clones fu-14, 15 and 16 were isolated; adherent cells were trypsinized, replated, treated with ConA and clone fu-13 was isolated.

Chromosome

analysis

Subconfluent populations of cells were grown on 100 mm plates in IO pg/ml colcemid, at 37”C, for 2.5 h.

und myogenesis

335

Cells were collected by trypsinization, centrifuged and the cell pellets were dispersed in 0.075 M KC1 and incubated 20 min at 37°C. Five drops of cold (4°C) acetic acid : methanol (1: 3) were added and the cells were centrifuged (450 g for 5 min), resuspended in 2 ml of cold acid : methanol and kept 1 h at 4°C. Treatment of the cell pellets in acid : methanol was repeated. The final pellets were resuspended in 1 ml acid : methanol, dropped onto cold glass slides and air-dried. Slides were incubated 1 h in 0.3 M NaCl, 0.03 M sodium citrate, pH 7.0, at 6o”C, then stained 1.5 h in 2% Giemsa in 0.02 M PBS, pH 6.8. One hundred welldefined chromosome complements were counted per cell sample.

In vitro transformation Colony formation in soft agar, uptake of [3H-2]deoxyglucose, adhesiveness and density-dependent inhibition of growth were assayed as reported [22]. Plasminogen-activator was determined by the case& proteolysis method [24]. Two hundred cells were plated/50 mm dish and incubated 8 days. The colonies were washed twice with warm Dulbecco’s PBS, pH 7.2, then overlayed with 3 ml of medium containing 0.4% agarose, 1.5% casein (Carnation non-fat dry milk) and 10% human serum. Cleared plaques in the overlay were scored 15 h later, the overlay was removed, the colonies were stained with 4% Giemsa and then counted. A minimum of 3 plates were done per group.

Expression of endogenous virus trunscriptase assay. Medium was changed in cultures 24 h prior to analysis for reverse transcriptase activity. Twenty ml of medium from two 100 mm culture plates were tested. The medium was removed from the mates. centrifueed seauentiallv at 1050 e for 5 min, ai IOOdO g for TO min and a<90000 g -(SW 27.1 rotor: 22000 mm) for 2 h at 4°C. The final oellets were resuspended in 50 mM KCI, 1.0 mM dithiothreitol (DTT) and IO mM Tris hydrochloride, pH 7.4 at 20°C. Reaction mixtures contained 50 mM KCl, 0.05% nonidet P40 (Particle Data Laboratory, Elmhurst, IL), 2.5 mM DTT, 1 mM MnCI,, 0.04 OD,,, units poly(C). Oligo(dG),,-,, (P-L Biochemicals), 22 PM [3H]dGTP (Amersham) and 10-50 ~1 of resuspended virus, in a total of 100 LLI and were incubated at 37°C for 60 min. Reactions were stopped by addition of 100 ~1 0.1 M sodium pyrophosphate in 20% trichloracetic acid (TCA); 20 pl containing 4 mg/ml yeast RNA was added to facilitate precipitation. The cold acid-insoluble precipitates were collected on 0.45 pm Millipore filters (presoaked in 5% TCA, 0.02 M pyrophosphate) and washed with 10% TCA, 0.02 M pyrophosphate. All reactions were done in duplicate.

Reverse

Moloney

Leukemia

Virus

Induced-Syncytia.

2~

105 cells were grown for 24 h in 2 ml of medium on 35 mm tissue culture dishes. The number of cells/ plate were then determined, the medium was removed and the cells were washed twice in PBS. Molonev Leukemia Virus (MuLV) was added at a multiplicity of 40, in 0.6 ml Dulbecco’s medium containing 12 pg/ ml DEAE dextran and the culture was incubated 1 h Exp Cell

Res 125 (1980)

336

Kaufman

et al. Tumor formation Athvmic female NIH Swiss nulnu mice (Harlan Industries) were injected subcutaneously with 3 X 106 cloned cells in 0.1 ml Dulbecco’s medium. These mice were maintained in germ-free isolators. Tumors were excised, minced and single cell suspensions prepared by dissociation in 0.25 % trypsin.

Quantitation

Fig. 1. Abscissa: time (days); ordinate: (left) nuclei in mvotubes/cm2 (O-- -0): (ripht) mUnits creatine kinase/mg protein‘(O-d,‘iiA). Myoblast fusion and creatine kinase activity. 2~ lo” E63 or fu- cells cloned from the L, line were plated in 50 mm dishes in 3 ml Dulbecco’s medium containing 10% horse serum, at 37°C in 10% CO, and high humidity. Medium was changed daily and duplicate cultures were assayed for myotube formation and creatine kinase activity. By day 5, more than 98% of the differentiating myoblast nuclei were within myotubes.

at 37°C. The virus was then removed and the plates incubated for 6 h in complete medium. The cells were then fixed in 70% methanol for 20 min and stained with fresh 4% Giemsa. The number of syncytia in at least 10 fields/plate and on a minimum of two plates were scored. This procedure was modified from that we previously reported [25].

Creatine kinase and myokinase

assays

To assay creatine kinase (CK) activity, reaction mixtures contained: 20 mM glucose, 10 mM magnesium acetate, 1 mM ADP, 20 mM AMP, 1 mM NADP, 10 mM cysteine, 15 mM creatine phosphate, hexokinase (1 U/ml), glucose-6-phosphate dehydrogenase (0.5 U/ ml) and 100 mM glycylglycine, pH 6.8. Conversion of NADP to NADPH at 30°C was monitored at 340 nm. CK values were corrected for the approx. 6% myokinase activity that persists under these conditions. Myokinase activity was determined in the absence of AMP and creatine phosphate. Cell samples were prepared as previously reported [22]. Cell protein was determined by the method of Oyama & Eagle [26]. Exp Cell Res 125 (1980)

of LETS protein

Iodination. Cell surface proteins were labelled with lz51 by a modification of the lactoperoxidase method described by Hynes [27]. 2x 105 cells were grown 36 h on 50 mm dishes, then washed thrice in warm PBS, pH 7.4. Two ml of PBS containing 1 mg of glucose, 0.2 mCi carrier-free Na’Y (Amersham) and 6.0 IU glucose oxidase (Worthington) were added per plate, at room temperature for 10 min. The reaction was effectively terminated by addition of KI to 10 mM and the cells were washed twice with PBS. Labelled cells were scraped from the plate, centrifuged (750 g for 5 min), resuspended in 0.1 ml, 62.5 mM Tris, pH 6.8, and sonicated at 4°C. Samnles were electrouhoresed on sodium dodecyl sulfate (SDS), 8-20% polyacrylamide gradient slab gels (0.7 mm thick) in Tris/glycine/SDS buffer [28] for 7 h at 10 mA. The gels were fixed, stained I221, dried on 3MM filter paper and autoradiographed onto Kodak XR-5 film. The developed film was scanned using an Ortec 43 10 densitometer and the percent of total radioactivity in the 250000 mol. wt peak was determined. Densitometric analyses of negatives prepared from the autoradiographs yielded the same results. ImmunojTuorescence. Cells grown on 1.1 cm dia glass coverslips coated with 0.1% gelatin were fixed in 3% formaldehyde in PBS, at 20°C for 20 min. The cells were extracted in acetone at -20°C for 7 min, washed thrice in PBS, drained, reacted with rabbit anti-human plasma cold-insoluble globulin (GIG) (purchased from M. Mosesson) in PBS, for 45 min at 37°C and extensively washed in PBS. The coverslips were then inverted over 20 ~1 FITC-conjugated goat antirabbit IgG (Miles) diluted 1 : 5 in PBS, for 30 min at 37°C. After extensive washings the coverslips were inverted onto a slide containing 5 ~1 glycerol : PBS (9 : l), sealed with Flo-texx (Lemer Laboratories) and stored at -20°C. Immunofluorescence was viewed with a Zeiss photomicroscope using epi-illumination and recorded using Kodak Ektachrome film exposed at an ASA of 320. The reactivity of anti-CIG antibody for LETS protein has been described by Bunidge [29].

RESULTS Developmental properties Creatine kinase activity closely parallels the differentiation of myoblasts into multinucleate myotubes. In the experiment depicted in fig. 1, fusion commenced 34 days after plating of 2~ 105 E63 cells and was es-

TranTformation

sentially complete by day 5. At that time more than 98% of the nuclei were within myotubes. Likewise, maximum creatine kinase activity is present by day 6. We have not detected creatine kinase activity in any of the non-fusing lines, although all nonfusing lines do exhibit myokinase activity comparable in quantity to that in the differentiating lines [22] and identical in electrophoretic mobility as indicated by starch gel electrophoresis (Kaufman, Loy & Bohn, unpublished observations). In addition, as previously shown for fu-1 cells, using SDSpolyacrylamide gel electrophoresis [22], we have not detected the increased myosin heavy chain content that is characteristic of fused myotubes in any of the non-fusing lines (data not shown). These analyses were done on both subconfluent and highly confluent cultures of the non-fusing lines. Expression

of endogenous

virus

We have previously shown that the fu-1 line but not the Ls cells fused into multinucleate syncytia when exposed to exogenous Moloney murine leukemia virus (MuLV) [25, 301. These viral-induced syncytia are both morphologically and functionally distinct from the myotubes that arise via spontaneous myoblast fusion, and they do not develop further characteristics of the differentiated state (unpublished data, [77]). Differential effects of various reagents on these two forms of cell fusion suggest that these multinucleate cells arise via different mechanisms [3 11. Although both L, and fu-1 cells can be infected by MuLV [25], the capacity of cells to be fused into syncytia seems to be associated with the expression of an endogenous RNA virus by these cells. The virus produced by fu-1 cells has been characterized as an endogenous type C rat retrovirus [32]. As shown in fig. 2, the production of viral particles having

and myogenesis

a-I

337

-

L8

-2

-3

-4

-6

i -8

,4

d0 -13

-14

-15 -16

2. Abscissu: cell line; ordinate: (left) reverse transcriptase activity (0) [3H]dGTP incorporated (cpm X 10m4);(right) syncytia/cmzX lo-* (B). Expression of endogenous virus by Ls cells and the non-fusing lines. Fusion of cells mediated by Moloney murine leukemia virus and reverse transcriptase activity of endogenous virus released into the medium were determined. Only those clones expressing the endogenous virus readily formed Moloney virusinduced syncytia.

Fig.

reverse transcriptase activity and the capacity to form MuLV-induced syncytia are parallel. Only those non-fusing lines producing the endogenous virus could be fused by MuLV. This may not be as evident for the fu-3 line; however, in the presence of MuLV, these cells formed large numbers of binucleate cells which were not scored as syncytia. All cell lines were routinely stored at -76°C in medium containing 20% horse serum and 10% dimethylsulfoxide. When retrieved and passaged, the fu-4, -6 and -8 lines liberated endogenous retrovirus. By the sixth consecutive passage, fu-13 and-14 also produced virus as measured by reverse transcriptase activity; virus production by fu-15 was detected after the 12th passage. No virus was produced by either the fu-16 or the Ls line or the E63 subclone even after Exp CellRrs

125(1980)

338 Table mice

Kaufman

et al.

1. Tumorigenicity

tion

LS

O/7

E63

O/l

fu-2 fu-3 fu-4 fu-5 fu-6 fu-7 fu-8 fu-9 fu-13 fu-14 fu-15 fu-16 fu-1 (virus)

nulnu

Days after injection tumor first evident

Virus produced by tumor explants pmoles r3H]dGTP incorporated/ IO6cells

O/6 l/7 517 (2) 616 717 717

617 (1) 717

616 417 (3) 818

717 517 (2) 6/7(l) z',::::' 617 (1)

517 8112 (4)

14; 7 13 12 7 9 7

i4 50.0 -

ti

57.8 7.8

;

2i.6 3.1 93.1 0.03

16 6 16 23 13 72 49

32.9 53.1 13.3

10.0 0.13 0.05

o/7

3 x 10” cells in 0.1 ml medium were injected subcutaneously. The numbers of animals developing tumors/ number mice injected are given. The numbers of mice that died before tumors were first evident are indicated in parentheses. One group of mice was injected with the 90000 g viral pellet obtained from the medium in which fu-l-cells were grown. Virus produced in 24 h by approx. 3x 10’ cells was injected per mouse. Animals in which tumors did not develop were kept up to 286 days. Retrovirus production was determined in the tumor explants. a Reverse transcriptase activity of the virus produced by the E63 derived tumor had optimal activity in magnesium, not in manganese, indicating that the viral enzyme was of mouse and not rat origin [32].

20 consecutive passages. Thus, 12 of the 13 non-fusing lines express an endogenous virus. Insofar as growth in vitro may select for (or induce) altered phenotypes, in all subsequent analyses cloned cells were retrieved from frozen stocks and utilized as soon as possible. Exp Cell

Table 2. Serum dependence and growth Growth in 10 and 1% serum

Tumor forma-

fu-1

in athymic

Res I.75 (1980)

Generation time (hours)

L8 fu- 1 fu-2 fu-3 fu-4 fu-5 fu-6 h-7

fu-8 fu-9 fu-13 fu-14 fu-1.5 fu-16

10% Serum

1% Serum

19.4 14.9 14.5 14.5 17.9 14.0 30.5 16.0 22.8 17.0 30.4 22.2 19.7 23.9

26.1 16.7 18.9 22.3 21.9 18.8 71.3 18.8 34.4 26.0 38.0 79.8 29.4 30.0

Saturation density 10%/l% 3.4 1.4 1.5 1.6 6.5 1.5 3.5 1.4 1.3 1.4 3.3 3.6

4.6 2.2

The doubling times of all lines were determined during exponential growth in 10 % and 1% serum by linear regression analysis (~~0.98). Relative saturation densities attained in 10% vs 1% serum indicate that ful-3, 5 and 7-9 do not maintain serum dependent growth requirements.

Tumor formation All non-fusing lines produced tumors when injected into athymic mice (table 1). These tumors were usually visually obvious within 2 weeks after the subcutaneous injection of 3 X 106 cells. Tumors developed considerably slower in mice injected with fu-15 and 16 cells; however, these “incubation” times do not appear related to the in vitro generation times of the respective cells (table 2). In our initial experiment with these mice, several animals died prior to development of visually evident tumors. This was subsequently determined to be due to a dietary deficiency which was readily remedied. Mice injected with L, or E63 cells or with endogenous fu-1 virus did not develop tumors even up to more than 8 months after injection (the one case in which E63 cells did form a tumor will be discussed). Two mice injected with fu-15 cells also did not form tumors. At least one tumor from mice

Trunsformation injected with each non-fusing line was surgically removed and explanted into tissue culture. All tumor-derived cells, except those from mice injected with fu- 13, - 15 and -16, liberated particles with reverse transcriptase activity. Notably, these clones did not originally express endogenous virus. Likewise tumors from mice injected with fu-4, -6, -8 and -14 produced less virus than those from mice injected with cell lines known to liberate the endogenous virus. One animal injected with 3x 10” E63 cells did develop a tumor after a prolonged “incubation” of 147 days. When explanted, these tumor cells did liberate virus; however, the reverse transcriptase activity of this virus had the divalent cation requirements characteristic of mouse and not rat retrovirus [32]. Chromosome analyses of all the tumor explants, including the E63-derived tumor, confirmed that these tumors were derived from the injected rat cells and were not of mouse origin. Growth in vitro As transformed cells often have reduced requirements for serum, we compared the growth of the non-fusing cells in 10 and 1% horse serum. 1 x lo5 cells were initially plated on 50 mm dishes in medium containing 10% serum. The medium was changed at 24 h and every day thereafter, and contained either 10 or 1% serum. Cells were collected by trypsinization from triplicate cultures and nuclei were counted as indicated in Materials and Methods. Generation times during the logarithmic phase of growth were determined by linear regression analysis of the data. As seen in table 2, the doubling times of several non-fusing lines in 10% serum were less than that of the parental L8 line, yet an equal number of defective lines grew slower. The generation time of most cell lines growing in 1%

and myogenesis

339

-17

gt 8

-6

7 -5

i

“t -4

-3

-2

I I 0

LB fu-15-14

-16 -13 -6

-4

-6 -7 -9

-0

cell line; ordinate: (left) [YH-2]deoxyglucose uptake (cpm x 10-3/mg proteinlmin); (right) rel. rate of uptake. Rates of hexose transport in the differentiating and non-fusing lines. 2~ lo5 cells were plated and grown on 50 mm dishes for 3 days. The cells were then washed with warm PBS and incubated at 37°C in 1 ml prewarmed PBS containing 1 &i/ml [3H-2]deoxyglucase (New England Nuclear) for 1, 5, 10, 20 and 30 min. Hexose uptake was linear under these conditions. The cells were solubilized and rates of uptake were determined as reported [22].

Fig. 3. Abscissa:

serum reflected their relative rates of growth in 10% serum; i.e., the ratio of generation times in 1% vs 10% serum was between 1.l and 1.6 for all lines except fu-6 and -14, the growth of which were more markedly slowed in 1% serum. The differences in the saturation densities attained in 10 and 1% sera were pronounced in the L, line and in the fu-6, -13, -14, -15 and -16 variants, and this is generally believed to be consistent with normal growth regulatory mechanisms. Although the saturation densities attained by lines fu-1, -2, -3, -4, -7 and -9 grown in 1% serum were consistently less than in 10% serum, these were always less than 1.6-fold differences and reflect a reduction in serum dependence characteristic of other transformed cells. Thus, the Exp CdRrs

125 (1980)

340

Kaufman

et al.

Table 3. Analysis of transformation Expression of endogenous VirUS”

L fu-1 (SP) fu-2 (SP) fu-3 (SP) fu-4 (P) fu-6 (P) fu-8 (SC) fu-5 (AP) fu-7 (AP) fu-9 (AP) fu-13 (ConA) fu-14 (ConA) fu-15 (ConA) fu- 16 (ConA)

Initially

Upon passage in vitro

+ + + + + + -

+ + + + + + + + + + +’ + -

Growth in 10% and I % serum* (Saturation density 10%/l %)

Growth in soft agarC (% cells forming colonies)

3.4

co.01

0

41.3 10.9 13.7

22.6 46.4 0 2.5 67.5 11.5 25.0 46.3 31.5 15.0 23.0 0 35.0

1.4 1.5

1.6 1.3 3.5 1.3 1.5 1.4

co.01

1.4

10.6 23.2 10.8 30.2 26.7

3.3 3.6 4.6 2.2

8.9
co.01
Proteolysi@ (% colonies hydrolyzing casein

Adhesiveness’ (% cells detached in EDTA at 30 min)

2-Deoxyglucose uptake’ Relative index

2 43 81 8 7 4 27 53 42 48 :

6.7 5.6 5.5 3.2 3.7 2.9 3.4 3.7 4.0

6 2

1.0

1.9 1.7 1.6 1.9

Those characteristics that are potential positive indicators of transformation are set in bold face. Cell lines are arranged according to the method of isolation (SP, serial passage; P, growth on non-sulfonated polystyrene; SC, spinner culture; AP, growth on agar-coated plates; ConA, treatment with ConA). a Virus-specific reverse transcriptase activity in medium in which cells were grown was determined as indicated in Materials and Methods. * Rel. saturation densities attained in 10 % and 1% serum. c Colony formation in 0.3% agar. lo* cloned cells were plated per 50 mm dish according to the method of MacPherson & Montagnier [76] and colonies were scored 7 days later. Lines not developing colonies were replated at up to 10’ cells/50 mm dish. d Colonies hydrolyzing casein in an agarose overlay formed clear plaques; this is indicative of elevated plasminogen activator [24]. e Reduced adhesiveness as determined by detachment in 4 mM EDTA. 2~ lo5 cells were plated per 50 mm dish and incubated 2 days. Cells were washed in Ca *+ , Mg2+-free Earle’s solution and incubated 30 min in the same buffer containing 4 mM EDTA. The numbers of cells detached were compared with the total numbers of cells obtained after trypsinization and agitation. f Rates of hexose transport determined by uptake of [3H-2]deoxyglucose as in fig. 3. The relative rates with respect to L, cells are given. 0 Quantitation of LETS protein by iodination and immunofluorescence. Cells were grown and labelled either with I*51 or anti-CIG antibody as indicated in Materials and Methods. The percent total radioactivity migrating in the 250000 mol. wt region upon SDS-polyacrylamide gel electrophoresis is given. The presence (+) or absence (-) of immunofluorescent fibers is indicated. ND, not done. h Tumor formation in NIH Swiss athymic mice.

growth of 8 of the 13 non-fusing lines appears to be relatively independent of the serum concentration. Elevated hexose transport is an early phenotypic change in cells transformed by sarcoma virus [33, 341 and this is evident in the fu-1 line [22]. The rates of hexose transport, as measured by the uptake of [3H-2]deoxyglucose, are also elevated to various degrees in all the non-fusing lines (fig. 3). Exp

Ceil Res 125 (1980)

There is a great deal of variability in the rates of hexose transport from line to line; however, these rates appear to fall into three classes (although this may be a reflection of the relatively small number of clones examined). Cells isolated by serial passage exhibit the highest rates of uptake (5.5 to 6.7-fold greater than L, cells); lines 4-8, isolated by three different methods, have intermediate (2.9- to 4.0-fold) elevated rates of

Transformation

and myogenesis

341

P LETS’ (fibronectin)

40-

Immunofluorescence

Tumor formation in athymic miceh

9.1 3.1

+ -

+

1.1

-

+

1.7 1.7 9.8 2.8 0.7 1.5 1.4 10.7 11.9 ND 12.1

+ + + + +

+ + + + + + + + + + +

121I

30-

20-

uptake. Lines 13-16, isolated by repeated exposures to ConA have less than 2-fold elevations in hexose transport and will be considered in the same class as L, cells in further analysis of these data as this was within the variation obtained with L8 cells in other experiments. We have likewise evaluated the non-fusing lines with respect to their capacity to grow in soft agar, their relative adhesiveness, as determined by EDTA-mediated detachment and the presence of elevated proteolytic activity as measured by the hydrolysis of casein by colonies of cells. This latter assay is based upon the premise that transformed colonies secrete an activator of serum plasminogen; as a consequence, plasmin hydrolyzes casein in the overlay producing a clear plaque. As hydrolysis was dependent upon serum and was completely inhibited by the protease inhibitors leupeptin and antipain, the cellular function we have measured appears analogous to the elevated plasminogen activator activities associated with many transformed cells (fig.

‘O- /

A

-------

0 4

5

/ ---6

---7

8

4. Abscissa: time (days); ordinafe: % colonies hydrolyzing casein. - Plasminogen activator production as determined by the casein hydrolysis assay [24]. 2X 102 E63 (O), fu-1 (0) or fu-7 (A) ceils were plated per 50 mm dish. The percent colonies hydrolyzing casein and forming clear plaques were determined in triplicate cultures after 15 h incubation with the casein-agarose overlays on the days indicated. Both 0.5 mM antipain and 0.25 mM leupeptin (Cl) included in the overlay on fu-1 or fir-7 cells, completely prevented hydrolysis (these levels of protease inhibitors were previously determined to have no effect on cell growth or viability). Further results on the other non-fusing lines are indicated in table 3. Fig.

4, table 3). Continued growth of L8 colonies for several additional days also did not result in casein hydrolysis. The presence of a large (250000 mol. wt) iodinatable cell surface glycoprotein (referred to as LETS or fibronectin) has been shown to be markedly reduced in many sources of transformed cells. This change is thought to be closely associated with alterations in the cellular cytoskeleton, adhesiveness and morphology that often accompany transformation (for reviews see [35, 361). To determine relative amounts of membrane LETS protein, cells were iodinated and the corresponding cell extracts were analyzed by gel electrophoresis and autoradiography. The relative amounts of lz51 in the 250000 mol. wt region were quantitated (fig. 5, table 3). These results were confirmed by immunofluorescent staining of LETS protein (fig. 6). Those lines with decreased Err, CdKr

125 (1980)

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fu-16 1413 9 8 7 6 5 4 3 2 1 LB

5. Gel electrophoresis and autoradiography of iodinated cell lines. All cell lines were grown and iodinated by the lactoperoxidase procedure and cell lysates were electrophoresed on 8-20 % polyacrylamide gradient slab gels and autoradiogranhed as indicated in Materials and Methods. Myosm (210000 mol. wt), lactoperoxidase (78000 mol. wt), actin (43000 mol. wt) and DNase I (31000 mol. wt) were included as markers. The autoradiographs were scanned and the percent total radioactivity migrating in the 250000 mol. wt LETS region was quantitated (table 3). Fig.

iodinatable LETS also did not demonstrate the fibrillar network seen in the single L, cells or in the fu-6, -13, -14, -15 and -16 lines (table 3). The partial reduction in iodinatable LETS on fu-1 cells was likewise paralleled by a partial reduction in fibers staining with anti-CIG. By all the criteria of transformation that we have measured, the L8 (and E63) cells were determined to be untransformed (table 3). The phenotypes of the non-fusing lines varied considerably. Only three lines, fu- 1, -2 and -9, were uniformly positive for all in vitro transformation criteria. Statistical analyses of the data in table 3 indicate that the initial expression of endogenous virus, decreased adhesiveness, serum independent growth, elevated hexose transport and

decreased LETS protein in the non-fusing lines are all significantly associated with one another (table 4). The association of these parameters of the transformed phenotype with tumor formation is not, however, statistically significant. The capacities of these cells to grow in soft agar and hydrolyze casein are clearly independent of each other and of any other in vitro property measured. As all but two of the nonfusing lines did hydrolyze casein, this parameter, as well as expression of endogenous virus, was most commonly associated with tumorigenicity. However, no single in vitro criteria of transformation was a totally reliable index of tumor formation. Although the number of clones isolated by any single technique was quite small, at least two patterns of the transformed phenotype emerge. There generally is a uniformity of the transformed phenotype in the lines isolated by any single method, although exceptions are noted in each. The transformed properties of the non-fusing clones isolated by serial passage and by growth on agar plates are quite consistent, although there was slight variability both within and between these groups. Likewise, the phenotypes of clones selected by their relative resistance to ConA were quite uniform, but differed considerably from the cells isolated by serial passage or by growth on agar. Fu-13, -14, -1.5 and -16 did not initially express endogenous virus, did not have markedly elevated hexose transport nor reduced LETS, adhesiveness or serum dependent growth, and as such represent a minimal association with transformed characteristics. On the other hand, these cells did grow in soft agar, did have elevated protease activity and did form tumors. It is interesting to note that fu-4 and -6, isolated by growth on non-tissue culture dishes, do not have similar phenotypes. Fu-6, for ex-

Transformation

Fig. 6. Detection of LETS fibers by indirect immunofluorescence. (A) L8; (B) fu-1; (C) fu-3; (D) fu-4; (E) fu-9; and (F) fu-13 cells were grown on glass coverslips and then reacted with rabbit anti-GIG antibody and fluorescein-conjugated goat anti-rabbit immunoglobulin. Fluorescent stained fibers were evident in the L, and fu-13 cells, but were absent on the fu-3, fu-4 and

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fu-9 cells. The reduction in fibers stained on fu-1 cells parallels the reduction in iodinatable LETS (fig. 5, table 3). Bar equals 20 pm. Further results on all the clones are indicated in table 3. In those lines where fibers are absent, diffuse staining, primarily over the nuclear region, was evident and recorded by prolonged exposures. Exp CdRe~l25

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Table 4. Statistical

analysis

of cell line phenotypes

Virus expressed upon passage Serum independent growth Growth in soft agar Elevated protease activity Decreased adhesiveness Elevated hexose transport Decreased LETS B Tumor development Muscle differentiation A

Virus initially expressed

Virus expressed upon passage

Serum independent growth

Growth in soft agar

Elevated protease activity

0.54 0.016” 1.0 1.0 0.025” 0.049” 0.016” 0.46 0.46

0.38 0.69 0.85 0.54 0.31 0.38 0.92 0.92

0.10 1.0 0.016O 0.007”
0.46 1.0 0.18 1.0 0.69 0.69

0.27 1.0 1.0 0.84 0.84

Values obtained for each characteristic of all cell lines were classified positive or negative based upon conformity to that expected for transformed cells (table 3). A, The probability that the association of any two parameters was not random was determined by 2x2 contigency tests and significant values at thepc0.05 level are indicated f’). B, The correlation coefficients (a-b)/(a+b) of each in vitro characteristic with tumorigenicity and differentiation are given.

ample, more closely resembles -14 than it does fu-4.

fu-13 and

Chromosome analysis

L, cells maintain a near diploid complement of chromosomes (rat 2N=42); however, the fu-1 line was found to be pseudotetraploid. Analysis of all other non-fusing clones within several passages of their isolation indicates that all these developmentally defective lines exhibit some degree of polyploidy (table 5). Although it is not possible to determine whether the original cloned cell of the lines that exhibit less than 100% polyploidy was in fact polyploid, it is likely that such was the case since the shift from diploid to polyploid in the L, cells is relatively rare and since the instability of the polyploid state is greater than that of the diploid. Furthermore, upon passage of fu-1 cells, we have isolated populations that are predominantly pseudodiploid. DISCUSSION The LB line of rat myoblasts was isolated from newborn outbred Wistar rats [37]. Exp Cell

Res 125 ( 1980)

Mutagens were not used in the selection of this particular line. These cells maintain their capacity to differentiate in vitro when serially propagated at low densities. Upon attaining confluency, L8 cells cease replication and spontaneously fuse; within 48 h thereafter more than 98% of nuclei are within myotubes. Elevated creatine kinase activity parallels fusion in these cells as well as in primary cultures of rat and chick myoblasts [38-40]. This enzyme is of the MM isotype characteristic of skeletal muscle [41, 421, and it is readily quantifiable; creatine kinase is thus a convenient and appropriate biochemical marker of skeletal muscle differentiation. We have likewise determined that it is the MM form of creatine kinase that appears in fused L, cells (Loy & Kaufman, unpublished data). None of the 13 non-fusing lines that we have isolated from the Ls line elaborate appreciable creatine kinase activity or increased levels of myosin heavy chain that are characteristic of differentiated skeletal muscle. That myokinase activity is prevalent in all these nonfusing Ls clones is of no surprise since maximum activity of this enzyme is also

Transformation and myogenesis

Decreased adhesiveness

Elevated hexose transport

Decreased LETS

Tumor development

0.049’ 0.016” 0.46 0.46

0.007” 0.69 0.69

0.61 0.61

1.0

present in single L, and E63 myoblasts. Analogous results have been obtained using the myogenic L6 line and non-fusing lines derived from it [M. L. Pearson, personal communication]. The relationship between cell fusion and the cessation of proliferative capacity during myogenesis is not certain, i.e., if cessa-

345

tion of replication precedes fusion or if myoblasts cease replication as a consequence of fusion (e.g., see [ll-13, 401). Nadal-Ginard [43] recently showed that a subclone of L, myoblasts ceases proliferating and becomes irreversibly committed to differentiate prior to fusion. This sequence of events is important when one considers the different types of non-fusing cells that may arise. Knudsen & Horowitz [44, 451 have recently provided evidence to support at least two distinct stages in myotube formation, aggregation and fusion. Earlier reports likewise proposed a discrete series of events in myoblast fusion [ll, 12, 381. If cessation of proliferation is dependent on fusion and if fusion requires cells attaining both the capacity to interact and the actual event of cell fusion, then two classes of replicating non-fusing cells might be expected: (1) those cells that have not attained the competence to aggregate and fuse would not exhibit any properties of differentiated myotubes and (2) cells with lesions in the actual fusion mechanism. As fusion

Table 5. Chromosome analysis % nuclei 2N

4N

8N

100 (38.9f 1 .O) 100 (38.1k3.2) fu- 1 fu-2 fu-3 fu-4 fu-5 fu-6 fu-7 fu-8 fu-9 fu-13 fu-14 fu-15 fu-16

81 (37.OkO.8)

33 (36.6& 1.3) 39(33.3+1.8) 65 (34.2& 1.7)

lOO(78.1~1.5) 100 (71.7k2.6) 100 (65.5k2.9) 19 (72.1 k2.0) 100 (72.3k3.5) 100 (68.7k2.2) 100 (72.2+ 1.9) 100 (72.8f2.4) 67 (71.4t2.6) 61 (64.2?2) 82 (65.1k3.8) 35 (67.7k3.3) 100 (63.323.1)

18 (130.21b8.4)

Chromosomes were prepared from each cell line as indicated in Materials and Methods and the numbers of chromosomes in 100 complements were scored. The percent cells having approx. 2, 4 or 8N chromosomes are given; the actual mean numbers f S.D. are in parentheses. 23-791817

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per se is not a prerequisite for expression of other parameters of the differentiated phenotype (e.g., see [46-50]), this latter class of cells would also be expected to have other characteristics of differentiated myotubes. Alternatively, if cessation of proliferation is coordinately regulated with or precedes the capacity to further differentiate, then all replicating, non-fusing myoblasts would not exhibit any parameter of the differentiated state. That none of our non-fusing lines, isolated by five different procedures, had elevated creatine kinase activity or myosin, supports this latter view. However, we cannot totally exclude the possibilities that either our isolation procedures or a lower frequency in generating cells defective in the fusion process per se, may have contributed to our selection for clones which do not express any characteristic of the differentiated phenotype. Since L, cells can be maintained as a permanent cell line, they may be considered to be transformed. However, this is probably not the case, for as previously discussed [22], these cells will not maintain their capacity to grow in vitro unless they are manipulated so that fusion is prevented. The normal course for these cells would be to terminate their capacity to replicate and to differentiate, thus ending their existence as a cell line. Thus “if left to their own devices”,, myogenic lines would not persist in vitro, and therefore they should be viewed as distinct from other permanent lines insofar as they maintain normal growth regulatory mechanisms. The developmentally defective clones of myoblasts, however, were shown to be transformed by several in vitro criteria often associated with oncogenic cells. In addition, all lines defective in differentiation produced tumors when injected into athymic mice. Thus, by both in vivo and in vitro t.r/,

Cdl

RPS 125 (IY8Oj

criteria, those cells that have lost normal growth control cannot permanently arrest proliferation and are unable to differentiate. In contrast, L8 and E63 cells retain normal growth control both in vitro and in vivo and maintain their capacity to differentiate. Analogously, differentiating lines of myoblasts derived from embryonal carcinoma cells are not tumorigenic [5 11, whereas nonfusing variants of the Ls line exhibit some properties of transformed cells [52, 531. That one athymic mouse did develop a tumor several months after injection of E63 cells and that this tumor produced mouse retrovirus, indicates that transformation of myogenic cells may occur in vivo as well. Nameroff et al. also suggested that mononucleate myogenic cells in a spontaneous mouse tumor may be tumorigenic because of their inability to suppress proliferation

WI. It is well documented that myoblast fusion takes place during the Gl portion of the cell cycle [13, 17, 181. Buckley & Konigsberg [18] and Konigsberg et al. [13] have analyzed this particular Gl phase during the differentiation of quail myoblasts and find that its duration is markedly increased and therefore distinct from the Gl of replicating myoblasts. Recent data indicate that when the Gl period of myoblasts is greater than 11 h there is a marked increase in the propensity of these cells to fuse and that cells must be in Gl at least 4 h for fusion to occur [ 131. We were therefore concerned that the developmental defect in our non-fusing lines might be related either to a decrease in their generation times and thereby a shortening of the Gl period or to the inability of these cells to regulate their replication times in response to media conditions. However, analysis of the growth of the lines in 10 and 1% sera revealed that several of the non-fusing lines have generation times

Transformation

greater than the differentiating lines and that the response of these cells to low serum concentrations is comparable, if not more pronounced, than that of differentiating L, cells (table 2). Whereas L8 cells do differentiate when grown in 1% serum, no evidence of this development (i.e., fusion, creatine kinase or increase myosin heavy chain) was found in any of the non-fusing lines grown under these conditions. Although some of the non-fusing cells do tend to pile up on one another when confluency is attained, all the lines do exhibit some degree of density related inhibition of growth that is presumably reflected in protracted Gl periods. We therefore feel that the developmental defect(s) in the non-fusing lines is (are) not directly related to an inability to establish extended Gl periods. That all the non-fusing lines exhibit some degree of polyploidy invites speculation as to the potential effect of this shift in ploidy on the origin of these cells. However, as selection methods may also influence the ploidy of transformed cells [54], the association of polyploidy with the developmental arrest of these cells may be fortuitous but certainly warrants further study. It is clear that once loss of growth control is established, polyploidy need not be maintained to sustain the inhibition of differentiation. Of the 13 non-fusing clones, six produced endogenous virus upon isolation. That six other lines eventually expressed this virus upon subsequent passages, suggests that some progression of events may be required for complete expression of the virus. The differentiated status of the cell may also regulate endogenous virus expression (e.g., see [55, 561). It is highly unlikely that production of virus was mediated by an infectious process since deliberate attempts to infect have been unsuccessful; in addition,

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this virus was shown to be endogenous to rat by molecular hybridization [32]. Whether expression of endogenous virus is causally related to the developmental defect in the non-fusing myoblasts is uncertain. It is clear that infection and transformation of chick and LR myoblasts with exogenous sarcoma virus precludes further differentiation [57-60]. Although endogenous retroviruses are generally thought to be non-oncogenic, these viruses may interact with the cell genome and evolve into transforming viruses [55]. In recent in vitro studies, non-transforming endogenous rat virus rescued host rat src genes and resulted in cell transformation and the production of transforming virus [61]. We have found an approximate loo-fold increase in the amount of src RNA in fu-1 cells compared to that in E63 cells [32] and this is consistent with the reports that expression of transforming viral genes inhibits myogenesis [57-60]. Alternatively, and as proposed by Fizman [58], integration of the viral genome at new sites might modulate the activity of genes essential for myoblast development. The possibility that the expression of the endogenous virus is a consequence of transformation must also be considered. Further evaluation of the role of the fu:l retrovirus in myogenesis is complicated by the fact that it is not readily infectious. However, this can be pursued using a transforming rat virus containing the endogenous transformation genes of fu- 1 cells and these studies are in progress. Our analyses reveal that no single in vitro parameter, often attributed to the growth of transformed cells, is totally consistent with the inhibition of differentiation or tumorigenicity of the non-fusing clones. As a consequence, we have no? been able to use any single in vitro criterion as an index of the frequency of formation of non-fusing vari-

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ants. The heterogeneity in the phenotypes of the non-fusing cells, especially the variation between lines isolated by different procedures, suggests that the mode of isolation is important in determining which transformed properties will be exhibited. This is supported by the finding of relatively homogeneous phenotypes within groups isolated either by serial passage, growth on agar or after repeated treatment with ConA. It is clear (tables 3, 4) that the expression of certain in vitro parameters, e.g., reduced adhesiveness, elevated hexose transport, decreased LETS, serum independent growth and expression of endogenous virus are closely associated with one another. Yet, it is probably equally significant that none of these criteria, and none of the other parameters measured, are reliable indices of the tumorigenicity of these lines. This contrasts with other suggestions that growth in soft agar [62-64], decreased LETS protein [65] or elevated plasminogen activator [66, 671 are in vitro hallmarks of tumorigenicity. Uniformities in transformed phenotypes between cells in culture may reflect common modes of selection or in vitro growth conditions. In addition, many characteristics of transformed cells may be more causally related to changes in cell morphology and/or adhesiveness rather than to alterations which are a direct consequence of transformation [e.g., see 68, 691. In skeletal myoblasts, alterations in growth properties and expression of other potentially useful measurements of transformation seem secondary to the loss of control of proliferation required for further myogenesis. It is this single characteristic that appears sufficient to preclude differentiation and promote tumor formation. As chemicals (e.g., 5-bromodeoxyuridine [70, 7 l] and 12-O-tetradecanoyl-phorbol13-acetate [72, 731) and transforming viruses [57E.rp CellRes

125 (1980)

601 that inhibit differentiation also promote proliferation further suggests that these are antagonistic processes, coordinately regulated during myogenesis. In addition, media rich in growth promoting substances (e.g., serum and embryo extract) tend to enhance growth and inhibit differentiation [74, 751, whereas deprivation of required constituents terminates proliferation and promotes differentiation [47]. We are grateful to MS Diane England for her excellent technical assistance and to Drs M. Pearson for sharing his results prior to publication, W. Troll for generously providing leupeptin and antipain, P. K. Y. Wong for supplying the MuLV, and P. Imrey for his heloful discussions regarding the statistical analvsis. This study was aided by aBasil O’Connor Starter Research Grant from the National Foundation March of Dimes and a grant from the Muscular Dystrophy Association.

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