A clonal human skeletal muscle cell line showing acid maltase deficiency (AMD) was established through the transfection of origin-defective SV40
DNA. The low acid a-glucosidase activity and glycogenosomes in this clone corresponded to AMD. This clone, in spite of loading glycogenosomes, was competent not only as to proliferation without contact inhibition but also as to myogenic differentiation to some extent. Dexamethasone promoted the formation by the transformant of multinucleated myotubes, which expressed acetylcholine receptors. The existence of glycogenosomes did not seem to affect the proliferationor differentiation of myoblasts. The aberrant acid a-glucosidase expressed in the transformed myogenic clone was shown to be biochemically identical to that in AMD fibroblasts. This transformant should be of great value for investigating the pathogenesis of AMD because of the possibility of supplying semi-permanently a uniform myogenic cell line expressing AMD. Key words: acid maltase deficiency myogenic cell line origin-defective SV40 DNA transfection dexamethasone MUSCLE & NERVE 14:245-252 1991
HUMAN ACID MALTASE=DEFICIENT MYOGENIC CELL TRANSFORMATION WITH ORIGIN-DEFECTlVE SV40: CHARACTERIZATION OF A CLONED LINE FUSAKO USUKI, MD, ITSURO HIGUCHI, MD, YASUKO SOEJIMA, MD, MASAKAZU HATTORI, PhD, IKURO MARUYAMA, MD, and MlTSUHlRO OSAME, MD
Acknowledgments: The authors thank Drs. I. Kamo and S. lshiura (National Institute of Neuroscience, NCNP. Kodaira, Tokyo, Japan) for their valuable comments.
man et al.6.7developed replication origin-minus simian virus 40 (ori-SV4O) DNA, which could be transfected into cells more easily and successfully without replication of viral virions. Through transfection of this mutant SV40 DNA, normalI9 and diseased' fibroblast clones, and normalg and diseased 7*1 human myogenic clones have been established so far. We applied this transfection technique involving ori-SV40 DNA to the transformation of a human acid maltase deficiency (AMD) muscle culture and could separate myogenic clones from among the transformed cells. In this study, we characterized this clone morphologically and biochemically. Using this uniform myogenic AMD clone, we investigated the influence of glycogenosomes on cell proliferation and differentiation, and the difference of acid a-glucosidase in fibroblasts from that in myogenic cells.
Address reprint requests to Dr. F. Usuki, Third Department of Internal Medicine, Kagoshima University School of Medicine, Kagoshima 890, Japan.
MATERIALS AND METHODS
Accepted February 15, 1990.
Source of Muscle. T h e biopsied muscle was ob-
CCC 0148- 639X/91/030245- 08 $04.00 0 1991 John Wiley & Sons, Inc.
tained from a 13-year-old girl whose acid a-glucosidase activity was decreased to 6% of normal,
F o r a human metabolic disorder, a culture system provides a useful means for investigating its pathogenesis. However, cultured muscle cells have some disadvantages: finite life spans, slow growth rates, and inevitable contamination by fibroblasts. Because of these disadvantages, the application of muscle cultures has been limited. Recently, a new biological technique, the transfection of viral DNA, was developed. This technique has enabled us to obtain cell clones that are uniform and immortal, and that show a rapid growth rate. Gluz-
From the Third Department of Internal Medicine, Kagoshima University School of Medicine, Kagoshima, Japan (Drs. Usuki, Higuchi, Soejima, Maruyama, and Osame) and Basic Research Laboratories, Toray Industries, Jnc.. Kanagawa, Japan (Dr Hattori)
AMD Transformant with Origin-Defective SV40
MUSCLE & NERVE
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245
with maltose as the substrate, with her and her parent's consent. Electron microscopy showed excessive free glycogen and glycogenosomes in cultured fibroblasts.
cells were fed every third day, and counted on days 2, 4, 6, and 8. There were triplicate dishes for each day and for each medium, and cell counts were all duplicated.
The primary culture was performed by means of the explant technique. T h e cultures were maintained in modified Eagle's medium supplemented with 15% fetal bovine serum, penicillin, streptomycin, and fungizone, under a humidified atmosphere of 5% CO, at 37°C. The outgrowing cells were harvested by trypsinization, and myoblast-rich secondary cultures were obtained by differential a d h e ~ i o nT. ~ h e growing cells were subjected to transfection.
Electron Microscopic Study. Specimens were obtained by scraping the cells with a Teflon policeman followed by 2 washings with phosphatebuffered saline (PBS). Each specimen was immediately fixed in 0.125 M cacodylate-buffered (pH 7.2) 2.5% glutaraldehyde and then processed for electron microscopy.
Muscle Culture.
Aliquots (0.5 mL) of Hepes-buffered saline containing 6 Fg of ori-SV40 DNA (mutant 8- 16, supplied by Dr. Gluzman) and 125 mmol/L Caf' were allowed to react with semiconfluent cells in a 6-well plate. After 2h at 37"C, the dish was washed with Trisbuffered saline (TBS) solution and then exposed to 20% glycerol in TBS for 3 min at room temperature. After rinsing with TBS, the cells were cultured in the growth medium for 4 days, and then trypsinized and replated on dishes (diameter 60 mm). After 2 weeks, a morphologically transformed focus was picked u p and replated on a separate 48-well plate in the growth medium. Sufficient growing cells were trypsinized and replated on dishes (diameter 60 mm). Such cloning was performed again after distinct colonies could be identified. Finally, 3 times-cloned lines were established.
Transfection and Cloning Procedures.
Analysis of SV40 DNA Integration in Chromosomal DNA of the Established Cell Lines. T h e integration
of SV40 DNA into the genomes of the established cell line was analyzed by Southern blot hybridization. Briefly, 20 kg of genomic DNA was digested with EcoRI or KpnI, and the resulting DNA fragments were electrophoresed on a 0.7% agarose gel, then transferred to nitrocellulose filters. Wild-type SV40 DNA was labeled in vitro by nicktranslation and then hybridized to cellular DNA. The filters were washed and then autoradiographed. Cultures were grown in 60-mm dishes seeded with lo5 cells per dish in 4 mL of the growth medium described above or serumfree medium (MEM, 5 kg/mL insulin, 5 pg/mL transferrin, and 5 ng/mL sodium selenite). T h e
Immunocytochemistry. For assaying cell-surface acetylcholine receptor antigens, fused multinucleated cells grown on a glass slide were incubated with a-bungarotoxin-horseradish peroxidase (aBTX) (provided by Dr. I. Kamo) for 2 h at 10°C. After washing twice with PBS, the binding sites for a-bungarotoxin on the cells were made visible by incubation in hydrogen peroxide and diaminobenzidine. Column Chromatography. The cells, suspended in an appropriate volume of distilled water, were disrupted by sonication for 10-s periods (Microson; Heat System- Ultrasonics Inc., NY; setting 2). After centrifugation at 10,000g for 15 min, the supernatant was applied to a 4 mL column (a disposable syringe from Terumo, Tokyo) of DEAE-cellulose equilibrated with 5 mM phosphate buffer, pH 7.0. The column was washed with 5 volumes of the same buffer and then eluted with a linear gradient of 0 to 0.5 M NaCl in the same buffer (37.5 mL each). Fractions of 2.5 mL were collected. DEAE-Cellulose
Acid (pH 4.0) and neutral (pH 6.5) a-glucosidase activities were determined with 4-methylumbelliferyl-a-~-glucoside(4MUG, Sigma Chemical Co., St. Louis, MO) or maltose as substrate as described previously.20 In kinetic studies, the concentration of 4MUG was varied up to 8 mR/I and that of maltose u p to 40 mM. Other lysosomal enzyme activities (a-galactosidase, a-mannosidase, a-fucosidase, (3-glucuronidase, P-galactosidase and N-acetyl-P-hexosaminidase) were determined in the same run with the corresponding 4MU compounds as substrates. Enzyme Assays.
Cell Growth.
246
AMD Transformant with Origin-Defective SV40
Immunoblotting. Confluent cultures were harvested and homogenized as described above, but in homogenization buffer comprising 5 mM potassium phosphate buffer, pH 7.0, containing 10 kg/
MUSCLE & NERVE
March 1991
mL leupeptin and 1 mM EDTA. Cell debris was removed by centrifugation for 15 min at 10,OOOg and the protein concentration was determined by the method of Lowry et ai.l3 For immunoblotting, 50 pL of a 1 : 1 suspension of concanavalin A/ Sepharose 4B in homogenization buffer was added to 300 pL (containing 500 pg of protein) of cell extract to bind acid a-glucosidase and other glycoproteins. The binding was carried out for 2 h at 4°C in a rotating tube. T h e Sepharose beads were collected by centrifugation and washed 5 times with homogenization buffer to remove nonspecifically bound proteins. The beads were finally resuspended in 100 pL of a protein solution (5% mercaptoethanol, 0.125 M Tris-HC1, pH 6.8, 4% SDS and 10% glycerol) and then the samples were heated at 85°C for 5 min. The solubilized proteins were separated on 10% polyacrylamide gels containing SDS according to Laemmli" and subsequently transferred to nitrocellulose filters. Polyclonal rabbit antihuman placental acid aglucosidase antibodies, which were monospecific, as determined by Western blotting, were used for the detection of acid a-glucosidase. Immune com-
plexes were visualized by means of the indirect peroxidase reaction. RESULTS The Establishment of Ori- SV40 Transformed Myo. genic Cell Line. T415 clone was selected from
among the established AMD cells, because the shape of this clone cells changed from polygonal to lengthened and began to fuse, though insufficiently, when cultured in the fusion medium (MEM + 2% FBS + 5 pg/mL insulin + 5 pg/mL transferrin + 5 ng/mL sodium selenite). The integration of SV40 DNA into the genome of T415 clone was examined by means of Southern blot hybridization analysis. Cos I cells, SV40-transformed simian cell line, were examined in parallel with our clone. The results show that the T,15 clone contains one or a few integrated SV40 DNAs (Fig. I), which indicated that this clone was transformed with ori-SV40 DNA. The growth curves for T415 clone are shown in Figure 2. In the growth medium, T415 continued to increase in cell number in spite of reaching confluence, whereas, in the serum-free medium
1 2 3 4
1 2 3 4
Kb
EcoRI
Kpnl
FIGURE 1. Southern blot analysis of SV40 sequences in cellular DNA. Cellular DNA (20 pg per lane) was digested with EcoRl or Kpnl and then analyzed by the Southern procedure as described in Methods section. Linear viral genomes are included for comparison in lane 1 (Cos I). Cellular DNA: lane 2, nontransformed case fibroblasts; 3, T,15; 4, T43b.
AMD Transformant with Origin-Defective SV40
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The Characteristics of the T415 Myoblastic Clone 0 T,15
0 T.36
growth medium ...... serum-free medium h
n
0 10 r
’
x
Y
w
c
0
0
2
4
6
8
Time ( day 1 FIGURE 2. Growth curves for SV40-transformed T415 clone. Solid lines denote cells grown in serum-containing medium and dotted lines cells grown in serum-free medium from day 2.
the growth of T,15 was clearly suppressed. Dexamethasone (400 ng/mL) triggered myogenic differentiation and led to the formation of niultinucleated myotubes (Fig. 3). On immunostaining of T415 with a-BTX, diffuse nonjunctional staining of the sarcolemma and some positive staining in the cytoplasm were observed (Fig. 4). These facts confirmed that T,15 was a myoblastic clone.
Electron Microscopic Study. Electron microscopy of the T,15 myoblastic clone showed the sequestration of glycogen in membrane-bound sacs (Fig. 5). Chromatographic Study. Figure 6 shows the elution profiles of a-glucosidase activities in cultured cell extracts on a DEAE-cellulose column. Both T,15 (Fig. 6A) and nontransformed case fibroblast (Fig. 6B) extracts gave a single small peak of acid a-glucosidase activity, that was eluted at 0.12 M NaCl. The activity of acid a-glucosidase in a normal fibroblast extract showed a single high peak, that was eluted at 0.15 M NaCl (Fig. 6C). In contrast, all of the three cell line extracts contained a large amount of neutral a-glucosidase activity, that was eluted at 0.26 M NaCl. Enzyme Activity. The activity of the acid a-glucosidase in the T415 myogenic clone amounted to only 26% of the normal level in fibroblasts, whereas that in nontransformed fibroblasts was 16% of the normal value. In contrast, neutral aglucosidase activity in both types of cells was essentially normal. Other lysosomal enzyme activities (a-galactosidase, a-mannosidase, a-fucosidase, Pglucuronidase, P-galactosidase, and N-acetyl-Phexosaminidase) in T,15 were 1.4 to %fold higher than those in normal fibroblasts (Table 1). The K,,L Values of a-Glucosidases. Using DE52-
FIGURE 3. Differentiated T415 clones. T415 clones were grown in the fusion medium supplemented with dexamethasone (400 ng/mL). Multinucleated fused cells are indicated by arrows (Bar = 0.1 mm).
248
AMD Transformant with Origin-Defective SV40
MUSCLE & NERVE
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FIGURE 4. lmmunostaining of a-bungarotoxin in cultured T415 myotubes. There is heavy staining of the sarcolemmal membrane region and, occasionally positive staining in the cytoplasm (Bar = 10 w).
purified enzymes, the kinetic characteristics of acid and neutral a-glucosidases in T, 15 were studied and compared with those in nontransformed or control fibroblasts (Table 2). The K,, values of the acid a-glucosidase for 4MUG and maltose in the T,15 clone were similar to those in nontransformed fibroblasts, but %fold higher than those in
~~
~
Table 1. Enzyme activities in cultured cells Activity (nmol MUihirng protein) Enzyme
T415
a-Glucosidase 29 8 Acid 136 0 Neutral a-Galactosidase 145 8 110 8 a-Mannosidase a-Fucosidase 137 3 p-Glucuronidase 124 7 734 9 a-Galactosidase 4006 0 N-Acetyl-phexosaminidase
Nontransformed fibroblast
controls. In contrast, the K,,, value of the neutral a-glucosidase for 4MUG was essentially identical to that in nontransformed or control fibroblasts. These results indicated that the characteristics of a-glucosidase isoenzymes in transformed myogenic T,15 cells were virtually identical to those in nontransformed fibroblasts. Immunoblotting Study. In immunoblotting experiments, a fibroblast extract of our AMD case showed no qualitative abnormality as to the processing of acid a-glucosidase. We found a 110 K precursor protein and a mature 70 K acid a-glu-
Normal fibroblast Table 2. The K, values of a-glucosidases in cultured cells
170 1084 50 7 76 5 44 4 47 1 523 1 1743 7
1112? 1043? 51 0 59 5 ? 56 0 63 1 ? 459 1 5 958 3 ?
Values are means i- SD wfth the numbers of samples
AMD Transforrnant with Origin-Defective SV40
* *
In
278(8) 324(8) 154(8) 22 9 (8) 26 0 (8) 26 1 (6) 1 2 4 4 (7) 506 6 (7)
parentheses
Acid a-glucosidase
Cell T,15 Nontransformed fibroblast Control fibroblast
Neutral a-glucosidase
4MUG (mM)
Maltose (mM)
4MUG (mM)
46 50
23 0 25 6
0 29 0 26
15
68
0 25
4MUG. 4-methylumbelliferyl-a-o-glucosfde
MUSCLE & NERVE
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249
FIGURE 5. Electron micrograph of T,15 clones. Membrane-bound particles of glycogen are present (arrows) (Bar
cosidase. T h e T,15 clone showed the same immunoblotting pattern as nontransformed fibroblasts (Fig. 7).
DISCUSSION
Acid maltase deficiency (AMD), glycogenosis 11, is a glycogen storage disease caused by a defect in the lysosomal enzyme, a-1,4-gl~cosidase.~ The pathophysiology of glycogen storage in AMD has not been elucidated completely. In the late-onset type of AMD, pathological and clinical manifestations are confined to skeletal muscle in spite of a defect of the enzyme in the liver, heart, and central nervous system, as well as in the skeletal muscle.'" The reason for this selective involvement of skeletal muscle in late-onset AMD is still unknown. This has tentatively been explained, ie, that the threshold of the enzyme activity on the accumulation of glycogen differs with each tissue, or that another glycogen-degrading enzyme is responsible for the intralysomsomal degradation of glycogen. T h e isoenzymes of acid a-glucosidase in
250
AMD Transformant with Origin-Defective SV40
=
1 pm).
various tissues and another glycogen-degrading enzyme have not yet been detected. Although uniform tissues are necessary for biochemical studies, biopsied muscle specimens, even muscle cultures, are contaminated by other tissues. And the quantities available for analysis are limited. Therefore, we applied the transfection technique involving ori-SV40 DNA to a human AMD muscle culture to obtain a uniform mass of a myogenic clone. In the present study, we could establish an acid maltase-deficient human myogenic clone, T, 15, through transfection of ori-SV40 DNA into the cellular genome. Though a mutant cell line that re-expressed normal enzyme activity after transformation was reported, this T,15 clone continued to express the mutant AMD phenotype observed in untransformed cells. T h e low activity of acid a-glucosidase was sufficient for the diagnosis of AMD. Electron microscopy confirmed the excessive free glycogen content and glycogenosomes. This clone, in spite of loading glycogenosomes, was competent not only as to great proliferation
''
MUSCLE 8. NERVE
March 1991
1
2
3
3c
8
6
20
v! 1c
Ip
I
0
0 U
30
FIGURE 7. lmmunoblotting of acid a-glucosidase. ConA-binding glycoproteins were isolated from cultured cell extracts and separated on a 10% polyacrylamide gel in the presence of SDS. After blotting onto a nitrocellulose filter, a specific antibody was applied, and then immune complexes were visualized by means of the indirect peroxidase reaction. All samples contained 0.5 mg of protein before the ConA treatment. Lane 1, nontransformed fibroblasts; 2, normal fibroblasts; 3, T,15.
.. ..
I
n 20
..-=>*
10
c 0
(P
al
0
u) (P
0
30
0
q
3
20
U 10
0
10
20
30
40
Frat. NO. FIGURE 6. DEAE-Cellulose column chromatography of a-glucosidases. (A) T415 cells, (B)nontransformed case fibroblasts, and (C) normal fibroblasts. Crude extract, containing approximately 3 mg (A), 4 mg (B), or 2.5 mg (C) protein, was applied. The collected fractions were assayed for 4MUG-degrading-aglucosidase activity at pH 4.0 ( 0 ) and pH 6.5 (o), respectively.
but also as to myogenic differentiation. From our results, a rare regenerative change in AMD musc1e2,3,14 does not seem to be due to the existence of glycogenosomes in AMD myogenic cells, if any. Though transformed cells retain certain differentiation traits to some extent, a few methods for more effective induction of differentiation have been reported. The transfection of temperaturesensitive oncogenic viruses’ or the SV40 large T-anti en gene coupled to a metallothionein promotor leads to an increase in the proliferative capacity with preservation of the capacity to differentiate physiologically. In our study, dexam-
F
AMD Transformant with Origin-Defective SV40
ethasone was effective for the induction of differentiation. Dexamethasone (400 ng/mL) promoted the formation by the ori-SV40-transformed myogenic T415 clone of multinucleated myotubes, which expressed the differentiation antigen, acetylcholine receptors. So far, different results as to the effect of dexamethasone on the fusion of myoblasts have been reported. Furcht et aL4 reported that dexamethasone (10 p M ) inhibited the fusion of normal myoblasts. In contrast, dexamethasone at concentrations of 40 to 400 ng/mL greatly accelerated myotube formation by myoid cells from the rat thymus. l o How dexamethasone facilitates the induction of myogenic differentiation of the SV40-transformed T, 15 clone remains to be elucidated. Using this uniform clone, the biochemical characteristics of acid a-glucosidase of AMD myoblasts were analyzed, ie, the K, value, and the elution pattern on DEAE-cellulose column chromatography and immunoblotting, and compared with those in normal fibroblasts. The results were as follows: (a) the T,15 clone had an aberrant acid a-glucosidase, the K,,, value of which was %fold higher than that in normal fibroblasts; (b) T,15 cell extracts gave a minor single acid a-glucosidase activity and a high peak of neutral a-glucosidase activity on a DEAE-cellulose column. The respective elution points were the same as those in normal fibroblast extracts; and (c) the processing pattern of the aberrant acid a-glucosidase was the same as that in normal fibroblasts. All the same results were obtained for nontransformed fibroblasts. Accordingly, we conclude that myoblasts do not have a different acid a-glucosidase isoenzyme from that in fibroblasts.
MUSCLE 8, NERVE
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251
This AMD myogenic clone is useful for cellular and molecular characterization of defects because of its uniformity, its capacity to proliferate greatly, and its ability to differentiate and to preserve a n aberrant enzyme. However, whether this clone constitutes a true “immortal” myogenic one re-
mains to be determined with longer-term cultures. For studying the pathophysiology of rare inherited human myopathies, this transfection proced u r e may be useful because we can readily obtain a uniform myogenic-differentiative clone with greatly increased proliferative capacity.
REFERENCES
1. Chou J Y : Human placental cells transformed by tsA mutants of simian virus 40: a model system for the study of placental functions. Pruc h’atl Acad Scz U S A 1978;75: 14091413. 2. Engel AG: Acid maltase deficiency in adult life. Morphologic and biochemical data in 3 cases of a syndrome simulating other myopathies. Excerpta Media International Congress Series No. 1 Muscle Dis lnt Proc Internat Con
DNA. The low acid a-glucosidase activity and glycogenosomes in this clone corresponded to AMD. This clone, in spite of loading glycogenosomes, was competent not only as to proliferation without contact inhibition but also as to myogenic differentiation to some extent. Dexamethasone promoted the formation by the transformant of multinucleated myotubes, which expressed acetylcholine receptors. The existence of glycogenosomes did not seem to affect the proliferationor differentiation of myoblasts. The aberrant acid a-glucosidase expressed in the transformed myogenic clone was shown to be biochemically identical to that in AMD fibroblasts. This transformant should be of great value for investigating the pathogenesis of AMD because of the possibility of supplying semi-permanently a uniform myogenic cell line expressing AMD. Key words: acid maltase deficiency myogenic cell line origin-defective SV40 DNA transfection dexamethasone MUSCLE & NERVE 14:245-252 1991
HUMAN ACID MALTASE=DEFICIENT MYOGENIC CELL TRANSFORMATION WITH ORIGIN-DEFECTlVE SV40: CHARACTERIZATION OF A CLONED LINE FUSAKO USUKI, MD, ITSURO HIGUCHI, MD, YASUKO SOEJIMA, MD, MASAKAZU HATTORI, PhD, IKURO MARUYAMA, MD, and MlTSUHlRO OSAME, MD
Acknowledgments: The authors thank Drs. I. Kamo and S. lshiura (National Institute of Neuroscience, NCNP. Kodaira, Tokyo, Japan) for their valuable comments.
man et al.6.7developed replication origin-minus simian virus 40 (ori-SV4O) DNA, which could be transfected into cells more easily and successfully without replication of viral virions. Through transfection of this mutant SV40 DNA, normalI9 and diseased' fibroblast clones, and normalg and diseased 7*1 human myogenic clones have been established so far. We applied this transfection technique involving ori-SV40 DNA to the transformation of a human acid maltase deficiency (AMD) muscle culture and could separate myogenic clones from among the transformed cells. In this study, we characterized this clone morphologically and biochemically. Using this uniform myogenic AMD clone, we investigated the influence of glycogenosomes on cell proliferation and differentiation, and the difference of acid a-glucosidase in fibroblasts from that in myogenic cells.
Address reprint requests to Dr. F. Usuki, Third Department of Internal Medicine, Kagoshima University School of Medicine, Kagoshima 890, Japan.
MATERIALS AND METHODS
Accepted February 15, 1990.
Source of Muscle. T h e biopsied muscle was ob-
CCC 0148- 639X/91/030245- 08 $04.00 0 1991 John Wiley & Sons, Inc.
tained from a 13-year-old girl whose acid a-glucosidase activity was decreased to 6% of normal,
F o r a human metabolic disorder, a culture system provides a useful means for investigating its pathogenesis. However, cultured muscle cells have some disadvantages: finite life spans, slow growth rates, and inevitable contamination by fibroblasts. Because of these disadvantages, the application of muscle cultures has been limited. Recently, a new biological technique, the transfection of viral DNA, was developed. This technique has enabled us to obtain cell clones that are uniform and immortal, and that show a rapid growth rate. Gluz-
From the Third Department of Internal Medicine, Kagoshima University School of Medicine, Kagoshima, Japan (Drs. Usuki, Higuchi, Soejima, Maruyama, and Osame) and Basic Research Laboratories, Toray Industries, Jnc.. Kanagawa, Japan (Dr Hattori)
AMD Transformant with Origin-Defective SV40
MUSCLE & NERVE
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245
with maltose as the substrate, with her and her parent's consent. Electron microscopy showed excessive free glycogen and glycogenosomes in cultured fibroblasts.
cells were fed every third day, and counted on days 2, 4, 6, and 8. There were triplicate dishes for each day and for each medium, and cell counts were all duplicated.
The primary culture was performed by means of the explant technique. T h e cultures were maintained in modified Eagle's medium supplemented with 15% fetal bovine serum, penicillin, streptomycin, and fungizone, under a humidified atmosphere of 5% CO, at 37°C. The outgrowing cells were harvested by trypsinization, and myoblast-rich secondary cultures were obtained by differential a d h e ~ i o nT. ~ h e growing cells were subjected to transfection.
Electron Microscopic Study. Specimens were obtained by scraping the cells with a Teflon policeman followed by 2 washings with phosphatebuffered saline (PBS). Each specimen was immediately fixed in 0.125 M cacodylate-buffered (pH 7.2) 2.5% glutaraldehyde and then processed for electron microscopy.
Muscle Culture.
Aliquots (0.5 mL) of Hepes-buffered saline containing 6 Fg of ori-SV40 DNA (mutant 8- 16, supplied by Dr. Gluzman) and 125 mmol/L Caf' were allowed to react with semiconfluent cells in a 6-well plate. After 2h at 37"C, the dish was washed with Trisbuffered saline (TBS) solution and then exposed to 20% glycerol in TBS for 3 min at room temperature. After rinsing with TBS, the cells were cultured in the growth medium for 4 days, and then trypsinized and replated on dishes (diameter 60 mm). After 2 weeks, a morphologically transformed focus was picked u p and replated on a separate 48-well plate in the growth medium. Sufficient growing cells were trypsinized and replated on dishes (diameter 60 mm). Such cloning was performed again after distinct colonies could be identified. Finally, 3 times-cloned lines were established.
Transfection and Cloning Procedures.
Analysis of SV40 DNA Integration in Chromosomal DNA of the Established Cell Lines. T h e integration
of SV40 DNA into the genomes of the established cell line was analyzed by Southern blot hybridization. Briefly, 20 kg of genomic DNA was digested with EcoRI or KpnI, and the resulting DNA fragments were electrophoresed on a 0.7% agarose gel, then transferred to nitrocellulose filters. Wild-type SV40 DNA was labeled in vitro by nicktranslation and then hybridized to cellular DNA. The filters were washed and then autoradiographed. Cultures were grown in 60-mm dishes seeded with lo5 cells per dish in 4 mL of the growth medium described above or serumfree medium (MEM, 5 kg/mL insulin, 5 pg/mL transferrin, and 5 ng/mL sodium selenite). T h e
Immunocytochemistry. For assaying cell-surface acetylcholine receptor antigens, fused multinucleated cells grown on a glass slide were incubated with a-bungarotoxin-horseradish peroxidase (aBTX) (provided by Dr. I. Kamo) for 2 h at 10°C. After washing twice with PBS, the binding sites for a-bungarotoxin on the cells were made visible by incubation in hydrogen peroxide and diaminobenzidine. Column Chromatography. The cells, suspended in an appropriate volume of distilled water, were disrupted by sonication for 10-s periods (Microson; Heat System- Ultrasonics Inc., NY; setting 2). After centrifugation at 10,000g for 15 min, the supernatant was applied to a 4 mL column (a disposable syringe from Terumo, Tokyo) of DEAE-cellulose equilibrated with 5 mM phosphate buffer, pH 7.0. The column was washed with 5 volumes of the same buffer and then eluted with a linear gradient of 0 to 0.5 M NaCl in the same buffer (37.5 mL each). Fractions of 2.5 mL were collected. DEAE-Cellulose
Acid (pH 4.0) and neutral (pH 6.5) a-glucosidase activities were determined with 4-methylumbelliferyl-a-~-glucoside(4MUG, Sigma Chemical Co., St. Louis, MO) or maltose as substrate as described previously.20 In kinetic studies, the concentration of 4MUG was varied up to 8 mR/I and that of maltose u p to 40 mM. Other lysosomal enzyme activities (a-galactosidase, a-mannosidase, a-fucosidase, (3-glucuronidase, P-galactosidase and N-acetyl-P-hexosaminidase) were determined in the same run with the corresponding 4MU compounds as substrates. Enzyme Assays.
Cell Growth.
246
AMD Transformant with Origin-Defective SV40
Immunoblotting. Confluent cultures were harvested and homogenized as described above, but in homogenization buffer comprising 5 mM potassium phosphate buffer, pH 7.0, containing 10 kg/
MUSCLE & NERVE
March 1991
mL leupeptin and 1 mM EDTA. Cell debris was removed by centrifugation for 15 min at 10,OOOg and the protein concentration was determined by the method of Lowry et ai.l3 For immunoblotting, 50 pL of a 1 : 1 suspension of concanavalin A/ Sepharose 4B in homogenization buffer was added to 300 pL (containing 500 pg of protein) of cell extract to bind acid a-glucosidase and other glycoproteins. The binding was carried out for 2 h at 4°C in a rotating tube. T h e Sepharose beads were collected by centrifugation and washed 5 times with homogenization buffer to remove nonspecifically bound proteins. The beads were finally resuspended in 100 pL of a protein solution (5% mercaptoethanol, 0.125 M Tris-HC1, pH 6.8, 4% SDS and 10% glycerol) and then the samples were heated at 85°C for 5 min. The solubilized proteins were separated on 10% polyacrylamide gels containing SDS according to Laemmli" and subsequently transferred to nitrocellulose filters. Polyclonal rabbit antihuman placental acid aglucosidase antibodies, which were monospecific, as determined by Western blotting, were used for the detection of acid a-glucosidase. Immune com-
plexes were visualized by means of the indirect peroxidase reaction. RESULTS The Establishment of Ori- SV40 Transformed Myo. genic Cell Line. T415 clone was selected from
among the established AMD cells, because the shape of this clone cells changed from polygonal to lengthened and began to fuse, though insufficiently, when cultured in the fusion medium (MEM + 2% FBS + 5 pg/mL insulin + 5 pg/mL transferrin + 5 ng/mL sodium selenite). The integration of SV40 DNA into the genome of T415 clone was examined by means of Southern blot hybridization analysis. Cos I cells, SV40-transformed simian cell line, were examined in parallel with our clone. The results show that the T,15 clone contains one or a few integrated SV40 DNAs (Fig. I), which indicated that this clone was transformed with ori-SV40 DNA. The growth curves for T415 clone are shown in Figure 2. In the growth medium, T415 continued to increase in cell number in spite of reaching confluence, whereas, in the serum-free medium
1 2 3 4
1 2 3 4
Kb
EcoRI
Kpnl
FIGURE 1. Southern blot analysis of SV40 sequences in cellular DNA. Cellular DNA (20 pg per lane) was digested with EcoRl or Kpnl and then analyzed by the Southern procedure as described in Methods section. Linear viral genomes are included for comparison in lane 1 (Cos I). Cellular DNA: lane 2, nontransformed case fibroblasts; 3, T,15; 4, T43b.
AMD Transformant with Origin-Defective SV40
MUSCLE & NERVE
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247
The Characteristics of the T415 Myoblastic Clone 0 T,15
0 T.36
growth medium ...... serum-free medium h
n
0 10 r
’
x
Y
w
c
0
0
2
4
6
8
Time ( day 1 FIGURE 2. Growth curves for SV40-transformed T415 clone. Solid lines denote cells grown in serum-containing medium and dotted lines cells grown in serum-free medium from day 2.
the growth of T,15 was clearly suppressed. Dexamethasone (400 ng/mL) triggered myogenic differentiation and led to the formation of niultinucleated myotubes (Fig. 3). On immunostaining of T415 with a-BTX, diffuse nonjunctional staining of the sarcolemma and some positive staining in the cytoplasm were observed (Fig. 4). These facts confirmed that T,15 was a myoblastic clone.
Electron Microscopic Study. Electron microscopy of the T,15 myoblastic clone showed the sequestration of glycogen in membrane-bound sacs (Fig. 5). Chromatographic Study. Figure 6 shows the elution profiles of a-glucosidase activities in cultured cell extracts on a DEAE-cellulose column. Both T,15 (Fig. 6A) and nontransformed case fibroblast (Fig. 6B) extracts gave a single small peak of acid a-glucosidase activity, that was eluted at 0.12 M NaCl. The activity of acid a-glucosidase in a normal fibroblast extract showed a single high peak, that was eluted at 0.15 M NaCl (Fig. 6C). In contrast, all of the three cell line extracts contained a large amount of neutral a-glucosidase activity, that was eluted at 0.26 M NaCl. Enzyme Activity. The activity of the acid a-glucosidase in the T415 myogenic clone amounted to only 26% of the normal level in fibroblasts, whereas that in nontransformed fibroblasts was 16% of the normal value. In contrast, neutral aglucosidase activity in both types of cells was essentially normal. Other lysosomal enzyme activities (a-galactosidase, a-mannosidase, a-fucosidase, Pglucuronidase, P-galactosidase, and N-acetyl-Phexosaminidase) in T,15 were 1.4 to %fold higher than those in normal fibroblasts (Table 1). The K,,L Values of a-Glucosidases. Using DE52-
FIGURE 3. Differentiated T415 clones. T415 clones were grown in the fusion medium supplemented with dexamethasone (400 ng/mL). Multinucleated fused cells are indicated by arrows (Bar = 0.1 mm).
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MUSCLE & NERVE
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FIGURE 4. lmmunostaining of a-bungarotoxin in cultured T415 myotubes. There is heavy staining of the sarcolemmal membrane region and, occasionally positive staining in the cytoplasm (Bar = 10 w).
purified enzymes, the kinetic characteristics of acid and neutral a-glucosidases in T, 15 were studied and compared with those in nontransformed or control fibroblasts (Table 2). The K,, values of the acid a-glucosidase for 4MUG and maltose in the T,15 clone were similar to those in nontransformed fibroblasts, but %fold higher than those in
~~
~
Table 1. Enzyme activities in cultured cells Activity (nmol MUihirng protein) Enzyme
T415
a-Glucosidase 29 8 Acid 136 0 Neutral a-Galactosidase 145 8 110 8 a-Mannosidase a-Fucosidase 137 3 p-Glucuronidase 124 7 734 9 a-Galactosidase 4006 0 N-Acetyl-phexosaminidase
Nontransformed fibroblast
controls. In contrast, the K,,, value of the neutral a-glucosidase for 4MUG was essentially identical to that in nontransformed or control fibroblasts. These results indicated that the characteristics of a-glucosidase isoenzymes in transformed myogenic T,15 cells were virtually identical to those in nontransformed fibroblasts. Immunoblotting Study. In immunoblotting experiments, a fibroblast extract of our AMD case showed no qualitative abnormality as to the processing of acid a-glucosidase. We found a 110 K precursor protein and a mature 70 K acid a-glu-
Normal fibroblast Table 2. The K, values of a-glucosidases in cultured cells
170 1084 50 7 76 5 44 4 47 1 523 1 1743 7
1112? 1043? 51 0 59 5 ? 56 0 63 1 ? 459 1 5 958 3 ?
Values are means i- SD wfth the numbers of samples
AMD Transforrnant with Origin-Defective SV40
* *
In
278(8) 324(8) 154(8) 22 9 (8) 26 0 (8) 26 1 (6) 1 2 4 4 (7) 506 6 (7)
parentheses
Acid a-glucosidase
Cell T,15 Nontransformed fibroblast Control fibroblast
Neutral a-glucosidase
4MUG (mM)
Maltose (mM)
4MUG (mM)
46 50
23 0 25 6
0 29 0 26
15
68
0 25
4MUG. 4-methylumbelliferyl-a-o-glucosfde
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FIGURE 5. Electron micrograph of T,15 clones. Membrane-bound particles of glycogen are present (arrows) (Bar
cosidase. T h e T,15 clone showed the same immunoblotting pattern as nontransformed fibroblasts (Fig. 7).
DISCUSSION
Acid maltase deficiency (AMD), glycogenosis 11, is a glycogen storage disease caused by a defect in the lysosomal enzyme, a-1,4-gl~cosidase.~ The pathophysiology of glycogen storage in AMD has not been elucidated completely. In the late-onset type of AMD, pathological and clinical manifestations are confined to skeletal muscle in spite of a defect of the enzyme in the liver, heart, and central nervous system, as well as in the skeletal muscle.'" The reason for this selective involvement of skeletal muscle in late-onset AMD is still unknown. This has tentatively been explained, ie, that the threshold of the enzyme activity on the accumulation of glycogen differs with each tissue, or that another glycogen-degrading enzyme is responsible for the intralysomsomal degradation of glycogen. T h e isoenzymes of acid a-glucosidase in
250
AMD Transformant with Origin-Defective SV40
=
1 pm).
various tissues and another glycogen-degrading enzyme have not yet been detected. Although uniform tissues are necessary for biochemical studies, biopsied muscle specimens, even muscle cultures, are contaminated by other tissues. And the quantities available for analysis are limited. Therefore, we applied the transfection technique involving ori-SV40 DNA to a human AMD muscle culture to obtain a uniform mass of a myogenic clone. In the present study, we could establish an acid maltase-deficient human myogenic clone, T, 15, through transfection of ori-SV40 DNA into the cellular genome. Though a mutant cell line that re-expressed normal enzyme activity after transformation was reported, this T,15 clone continued to express the mutant AMD phenotype observed in untransformed cells. T h e low activity of acid a-glucosidase was sufficient for the diagnosis of AMD. Electron microscopy confirmed the excessive free glycogen content and glycogenosomes. This clone, in spite of loading glycogenosomes, was competent not only as to great proliferation
''
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March 1991
1
2
3
3c
8
6
20
v! 1c
Ip
I
0
0 U
30
FIGURE 7. lmmunoblotting of acid a-glucosidase. ConA-binding glycoproteins were isolated from cultured cell extracts and separated on a 10% polyacrylamide gel in the presence of SDS. After blotting onto a nitrocellulose filter, a specific antibody was applied, and then immune complexes were visualized by means of the indirect peroxidase reaction. All samples contained 0.5 mg of protein before the ConA treatment. Lane 1, nontransformed fibroblasts; 2, normal fibroblasts; 3, T,15.
.. ..
I
n 20
..-=>*
10
c 0
(P
al
0
u) (P
0
30
0
q
3
20
U 10
0
10
20
30
40
Frat. NO. FIGURE 6. DEAE-Cellulose column chromatography of a-glucosidases. (A) T415 cells, (B)nontransformed case fibroblasts, and (C) normal fibroblasts. Crude extract, containing approximately 3 mg (A), 4 mg (B), or 2.5 mg (C) protein, was applied. The collected fractions were assayed for 4MUG-degrading-aglucosidase activity at pH 4.0 ( 0 ) and pH 6.5 (o), respectively.
but also as to myogenic differentiation. From our results, a rare regenerative change in AMD musc1e2,3,14 does not seem to be due to the existence of glycogenosomes in AMD myogenic cells, if any. Though transformed cells retain certain differentiation traits to some extent, a few methods for more effective induction of differentiation have been reported. The transfection of temperaturesensitive oncogenic viruses’ or the SV40 large T-anti en gene coupled to a metallothionein promotor leads to an increase in the proliferative capacity with preservation of the capacity to differentiate physiologically. In our study, dexam-
F
AMD Transformant with Origin-Defective SV40
ethasone was effective for the induction of differentiation. Dexamethasone (400 ng/mL) promoted the formation by the ori-SV40-transformed myogenic T415 clone of multinucleated myotubes, which expressed the differentiation antigen, acetylcholine receptors. So far, different results as to the effect of dexamethasone on the fusion of myoblasts have been reported. Furcht et aL4 reported that dexamethasone (10 p M ) inhibited the fusion of normal myoblasts. In contrast, dexamethasone at concentrations of 40 to 400 ng/mL greatly accelerated myotube formation by myoid cells from the rat thymus. l o How dexamethasone facilitates the induction of myogenic differentiation of the SV40-transformed T, 15 clone remains to be elucidated. Using this uniform clone, the biochemical characteristics of acid a-glucosidase of AMD myoblasts were analyzed, ie, the K, value, and the elution pattern on DEAE-cellulose column chromatography and immunoblotting, and compared with those in normal fibroblasts. The results were as follows: (a) the T,15 clone had an aberrant acid a-glucosidase, the K,,, value of which was %fold higher than that in normal fibroblasts; (b) T,15 cell extracts gave a minor single acid a-glucosidase activity and a high peak of neutral a-glucosidase activity on a DEAE-cellulose column. The respective elution points were the same as those in normal fibroblast extracts; and (c) the processing pattern of the aberrant acid a-glucosidase was the same as that in normal fibroblasts. All the same results were obtained for nontransformed fibroblasts. Accordingly, we conclude that myoblasts do not have a different acid a-glucosidase isoenzyme from that in fibroblasts.
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This AMD myogenic clone is useful for cellular and molecular characterization of defects because of its uniformity, its capacity to proliferate greatly, and its ability to differentiate and to preserve a n aberrant enzyme. However, whether this clone constitutes a true “immortal” myogenic one re-
mains to be determined with longer-term cultures. For studying the pathophysiology of rare inherited human myopathies, this transfection proced u r e may be useful because we can readily obtain a uniform myogenic-differentiative clone with greatly increased proliferative capacity.
REFERENCES
1. Chou J Y : Human placental cells transformed by tsA mutants of simian virus 40: a model system for the study of placental functions. Pruc h’atl Acad Scz U S A 1978;75: 14091413. 2. Engel AG: Acid maltase deficiency in adult life. Morphologic and biochemical data in 3 cases of a syndrome simulating other myopathies. Excerpta Media International Congress Series No. 1 Muscle Dis lnt Proc Internat Con
