JOURNAL OF CELLULAR PHYSIOLOGY 145:42&433 (1990)

Characterization of Flat Revertants Isolated From Cells Transformed by Abelson Murine Leukemia Virus (Ab-MuLV) TAKASHI OKA,* MASUO YUTSUDO, HIROKAZU INOUE, FUJITOHICUCHI, AND AKIRA HAKURA Department of Pathology, Kochi Medical School, Kohasu, Nankoku, Kochi 78 1-51 (J.O.) and Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka 565 (M.Y., H.I., F.H., A.H.), japan

Transformed Fisher rat fibroblast cell lines by Abelson rnurine leukemia virus frequently revert to the normal phenotype in usual culture conditions. Molecular biological analysis of three revertant clones isolated from the transformants showed that their morphological reversions were due to inactivation of the v-abl oncogene at multi le steps including transcription, translation or v-abl protein kinase activity itse f without any change in structural gene expression of helper virus. These findings suggest the existence of a specific rnechanism(s) for elimination of the v-abl oncogene by segregation, mutation, or gene rearrangement in these cells.

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Abelson murine leukemia virus (Ab-MuLV) has u-abl oncogene which is known to encode a fused protein with a molecular weight of approximately 120,000 daltons (~12Og"g-~~'). Ab-MuLV transforms both lymphoid cells and fibroblasts in cell culture and induces a rapid B-cell lymphoma in vivo. In the course of characterization of rat fibroblast cell lines transformed with Ab-MuLV, we found that these transformants frequently revert to the normal phenotype in standard culture conditions. In this paper we report the isolation and characterization of three flat revertants from these transformed cells. In these revertants, multiple steps involved in expression of the u-abl-oncogene were found to be responsible for their phenotypic reversions. Analysis of the molecular mechanism of this process of reversion should be very useful for understanding the state or behavior of exogenous genomes in eukaryotic cells. MATERIALS AND METHODS Cell lines The rat fibroblast cell lines used in this study were routinely passaged in Dulbecco-modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS). The hypoxanthine-guanine phos horibosyl transferase deficient (HGPRT-) fibroblast ce 1line, No. 7, was originally derived from the Fisher rat fibroblast F2408 line (Freeman et al., 1975). Clone No. 7 showed typical properties of an untransformed cell line, including a low saturation density and anchorage-dependent growth as judged by its inability to grow in soft agar. Clones Abl-7-1, Abl-7-2, Abl-7-3, Abl-7-5 were transformants of clone No. 7 obtained with Abelson murine leukemia virus (Ab-MuLV[Hixl).The spontaneous flat revertants used in this work, Rev-2, Rev-3, and Rev-5, were isolated from Abl-7-2, Abl-7-3, and Abl-7-5, re-

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0 1990 WILEY-LISS. INC.

spectively. The reversion rates of these transformed clones ranged from to Colony formation in soft agar Cells were inoculated into 0.33% upper agar on 0.5% basal agar (both in DMEM containin 10% FCS). After 3 weeks culture, colonies of more t an 0.125 mm in diameter were counted. Focus formation assay Inocula of 2.0 X lo5 No. 7 cells in 6 cm dishes were incubated overnight at 37°C. The cultures were then treated with polybrene (2 tJ.g/ml)for 30 min, and 0.2 ml of Ab-MuLV preparations were added. After adsorption at 37°C for 1hr, the cultures were covered with DMEM containing 5% FCS. After 3-5 days, the medium was changed to DMEM containing 2% FCS, and after incubation for 10 days transformed foci were counted. Southern blot analysis Kpn-I digests of DNAs were electrophoresed on 0.8% agarose gels and transferred to nitrocellulose filters as described (Southern, 1975; Roberts and Axel, 1982). The filters were prehybridized for 12 hr at 39°C in 50% formamide, 4xSSC (600 mM NaCl, and 60 mM Na citrate), 5xDenhardt (0.1% Ficoll-400,0.1% BSA, 0.1% polyvinyl pyrrolidone), 0.2% SDS, and 50 pg/ml denatured herring sperm DNA. Hybridization was performed for 24 hr at 39°C in the above buffer. The pAbl-2 plasmid, which consisted of a 2.3 kb Bgl-I1 fragment of v-abl oncogene in the pBR322 plasmid, labeled with a

a

Received October 27, 1989;accepted July 20, 1990. *To whom reprint requests/correspondence should be addressed.

REVERTANTS OF Abl-ONCOGENE TRANSFORMED CELL LINES

multi-prime labeling system (Amersham Oligolabeling kit), was denatured and used at a concentration of 10 ng/ml. After hybridization, the filters were washed three times with 2XSSC, 0.1% SDS at room temperature, and then three times with O.lxSSC, 0.1% SDS at 50°C. Northern blot analysis Total cellular RNA was extracted by the guanidiurd cesium chloride method (Glisin et al., 1974).Samples of 10 Fg of each RNA were denatured with glyoxal and dimethylsulfoxide and electrophoresed in 1.4% agarose gel as described by Maniatis et al. (Maniatis et al., 1982). After electrophoresis, RNA species were blotted onto a Pall-Biodyne membrane filter according to the directions of the su lier (PALL Corp.). The pAbl-2 plasmid, radiolabele y the same method as for Southern blot analysis, was used to probe the RNA blots for v-abl mRNA. Conditions for reh bridization, hybridization, and washing of the R A b ots were the same as for Southern blotting (high stringency conditions). Radioactive labeling of proteins Inocula of 5x lo6 cells were plated into 10 cm dishes. The followin da the medium was changed to methionine-free D E containing 5% dialyzed FCS. After starvation for 3 hr, the cells were radiolabeled with 250 FCi of 35S-methionine (Amersham, specific activity 1,000-1,300 Cilmmol) for 4 hr at 37°C in 2.5 ml of methionine-free DMEM. The labeled cells were washed twice with cold PBS (-1 and lysed by incubation in RIPA buffer (10 mM Tris-C1 [pH 7.21,150 mM NaC1, 1% Triton X-100, 1%sodium deoxycholate, 0.1% SDS, 100 KIU/ml Trasylol) for 20 min at 4°C. The lysate was centrifuged a t 10,000 rpm for 30 rnin at 4"C, and the supernatant was stored at -70°C until use. Immunoprecipitation of P120 For immunoprecipitation, sam les of 6 ~ 1 cpm 0 ~ of cell lysates were incubated for 1 r at 4°C with 5 to 15 ~1 of normal rat serum, SD rat anti-Molony MSV serum, or goat anti-Gross MuLV serum. Immune complexes were bound to 4 mg of protein A-Sepharose (CL4B; Pharmacia, Freiburg) and washed 4 to 5 times with RIPA buffer, once with KC1-Triton buffer [50 mM Tris-C1 (pH 7.3, 1 M KC1, 1% Triton X-100, 100 KIU/ml of Trasylol (Boehringer Manheimll and once with Tris-buffer (50 mM Tris-C1, pH 7.4). The washed immunoprecipitates were extracted with 35 ~1 of sample buffer (4% SDS, 0.125 M Tris-C1, pH 6.8, 20% glycerol, 10% 2-mercaptoethanol, 0.004%bromophenol blue) for 2 min at 100°C. Then the extracts were separated by 10% SDS-PAGE (Laemmli, 1970). The gels were stained, destained, and treated with 1 M sodium salicylate for 30 min at room temperature (Chamberlain, 1979). They were then dried and exposed to X-ray film (X-Omat AR, Kodak, Stuttgart). abl-Kinase assay Immunoprecipitation was carried out in excess normal rat serum (10 p1) or SD-rat anti-Gross-MuLV serum (10 pl) with unlabeled cell lysates (100 pg protein in RIPA buffer) for 60 min at 4°C.The immunoprecipitates were collected with protein-A Sepharose (Pharmacia CL4B), and washed four times with RIPA

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buffer and once with kinase buffer (20 mM Tris-C1, pH 8.0; 10 mM MgC12).They were then suspended in 20 ~1 kinase buffer with 5 pCi [32Pl-gamma-ATP (Amersham, 3,000 Ci/mmol) and incubated for 30 min at 30°C. After addition of 15 p1 of 4X concentrated sample buffer and boiling for 2 min at 100°C, the samples were centrifuged for 2 min at 10,000 rpm and supernatants were separated on 10% SDS-polyacrylamide gel. Phosphorylated polypeptides were detected by autoradiography.

RESULTS Clones Abl-7-1, Abl-7-2, Abl-7-3, and Abl-7-5 were isolated from transformed foci of clone No. 7 induced by Abelson murine leukemia virus (Ab-MuLV[Hixl).During culture in vitro, small populations of all these transformants reverted s ontaneously to the normal to phenotype of mor hology ;eversion rate: Several clones o flat revertants were isolated from each transformed line and stable clones of flat revertants; Rev-2, Rev-3, and Rev-5, from transformants Abl-7-2, Abl-7-3, and Abl-7-5, respectively, were used for further studies. The morphologies of transformants and revertants are shown in Figure 1. Transformants were spindle-shaped, appeared refractile by phase contrast microscopy, and grew three-dimensionally. In contrast, revertants, like the parent No. 7 cells, were flat cells that did not pile up in liquid culture. More than 50%of the transformants grew without anchorage dependence and formed colonies in soft agar, whereas less than 0.002% of the revertants formed colonies in soft agar (Table 1). All the transformants produced virus, measured by focus formation assay, Abl-7-1 being a particularly high producer, whereas the revertants did not produce any focus forming viruses (Table 2). These revertants also lost several other properties associated with transformation induced by Ab-MuLV infection, including a small cell size, high growth rate, high saturation density, and ability to grow in medium containing a low concentration of serum. In these respects, the revertants recovered normal phenotype. Next we investigated the expression of the provirus genome at the transcriptional and translational levels in a series of Abelson murine leukemia virus transformed Fisher rat cell lines and their revertant cell lines. We examined whether the v-abl gene was still integrated into genomic DNA in revertants and whether gross rearrangements of the v-abl gene had occurred in these cells by Southern blot analysis under stringent conditions (Fig. 2). Several copies of the viral oncogene were found to be retained into cellular DNA in revertants; however, copy numbers of v-abl oncogene were reduced in every revertants compared with the intensity of c-abl oncogene. Then the expressions of the v-abl oncogene at the transcriptional level in the transformed and revertant cell lines were analyzed by Northern blot hybridization (Fig. 3). All the transformants were found to express high levels of u-abl RNA exce t the Abl-7-2 cell line, in which expression of the u-al f1 gene was low and the u-abl transcript size was slightly enlarged. The revertant cell lines showed various types of gene expression: Revd cells expressed normal u-abl RNA in as large amount as the parent transformed cell line, Abl-7-5;

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OKA ET AL.

430

Fig. 1. Morphological appearance of transformants and revertants by phase contrast microscopy. 1: No. 7. 2: Abl-7-1. 3 Rev-2. 4 Abl-7-2. 5: Rev-3. 6 Abl-7-3. 7: Rev-5. 8: Abl-7-5.

TABLE 1. Colonv formation of transformants and revertants in soft AGAR Cells

10’

ceIls/plate

Inoculated cell No. lo4 cells/plate

10% cells/plate

Efficiency (%)

Abl 7-2 Abl 7-3

Abl 7-5 Rev-2 Rev-3 Rev-5 No. 7 ‘NO. of colonies per plate in soft agar

Rev-3 cells did not express v-abl RNA. Rev-2 cells expressed a different size of transcripts from that in the parent transformant, Abl-7-2, which expressed v-abl transcripts of slightly larger molecular size than norma1 transcripts of u-abl gene.

Next, translation of the Ab-MuLV genome in transformants and revertants was investigated by immunoprecipitation assay. As the genome of Ab-MuLV contains v-abl fused to a portion of the gag gene of MuLV (Reynoldset al., 1978), antibodies t o structural proteins

431

REVERTANTS OF Abl-ONCOGENE TRANSFORMED CELL LINES TABLE 2. Virus production of transformants and revertants Dilution of culture supernatant Cells

Xl00

Abl-7-1 Abl-7-2 Abl-7-3 Abl-7-5 Rev-2 Rev-3 Rev-5 No. 7

(250,120) (5030) (770, 1,190) (0.0)

(om

x10--'

x10-2

x10-3

FFU/ml

-

(680,840)

(150,180)'

6.1 X lo5 2.6 x 1 0 7 4.2 X 10'

(60,10)

(2,O) (740, 1,060) (0.0) (0.1)

(0,O) (0.0)

(0,O) (0.2)

(2,O)

(OD)

(4030) (0,O) (0,O) (090)

(0.0)

-

2.4 x lo4 0 0 0

-

-

0

'Transformed foci per plate

10.0-

4.9-

4

c-abl

4

v-abl

Fig. 2. Detection of v-abl provirus genome by Southern blot analysis. Genomic DNA from transformed and revertant cell lines was digested with Kpn-I, which cleaved viral DNA in the long terminal repeat (LTR). The digests were electro horesed on 0.8% agarose gel, transferred to a nitrocellulose membrane filter, and hybridized with the h"labe1ed 2.3 kb BglII fragment of the v-abl gene cloned in a pBR322 plasmid.

of MuLV can precipitate gag-abl pol rotein by reacting with its gag constituent (Reyno ds et al., 1978; Witte et al., 1978). Therefore, antibodies of rat antiMolony sarcoma virus and of goat anti-Gross leukemia virus were used to detect the product of the u-abl gene. The gag-abl product of the most widely used strain of Ab-MuLV has a molecular weight of approximately 120,000daltons (P120gQg-"*'),which has been shown to be the sole product of the Ab-MuLV genome (Baltimore et al., 1980). Expression of P120 was observed in all Ab-MuLV transformed cell lines and precursor proteins Pr65gQg,Pr7tigQg,Pr80'"", and P30gag,corresponding to the helper virus, Mo-MuLV(Hix), were also detected (Fig. 4). However, in the Abl-7-2 cell line, P120 was slightly shifted toward a higher molecular weight, in correspondence with enlargement of the u-abl RNA in this line. The extents of expression of P120 and the helper virus precursor proteins in the Rev-5 line were similar to those in the parent transformant Abl-7-5,but P120 was not detected in the Rev-2 and Rev-3 cell lines,

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in spite of normal expression of the helper virus precursor proteins. We examined whether the u-abl roduct expressed, P120gQg-ab',was functionally active y assay of protein kinase (Fig. 5). Autophosphorylation of P120gag-ab' was detected in all transformants at high levels except in the Abl-7-5 cell line, in which the activity was lower. On the other hand, no autophosphorylation of P120gQg-ab'detectable in any of the revertants. On super-infection with ecotropic Ab-MuLV(eco) all the revertant cell lines were re-transformed. Transformed foci were detected in Rev-5 cell line, however exact number of foci was not countable with the difficulty of morphology to distinguish the foci from background cells (Table 3). DISCUSSION Phenotypical reversion from transformed cells might be induced by the following mechanisms. First, the viral oncogene might be inactivated or altered by

E

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3256

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1106

-

mutation, or deleted. Second, expression of the viral oncogene might be reduced at the transcriptional or translational level by activation of a suppressor gene or in some other way. Third, cellular targets of viral oncogene products that are essential for the ex ression of the transformed phenotype might be alterei. In the case of the Rev-3 cell line, integration of the v-abl gene into genomic DNA was retained, as shown by Southern hybridization analysis, but no transcriptional or translational roducts or u-abl specific protein kinase activity were (Petected, suggesting that in this line phenoty ic reversion was due to switch off of the expression o the u-abl gtne at the transcriptional level. However, the gag or enu gene products of the helper virus, Mo-MuLV(Hix), were expressed in this revertant, indicating that the transcriptional blockage was specific to the Ab-MuLV(Hix) gene; thus, the phenotypic reversion may have been induced by mutation or rearran ement in the regulatory sequences of the Ab-MuL (Hix) gene, or by activation of a specific suppressor gene. The former possibility seems the more likely because the transformed phenotype could be recovered by introduction of an intact v-abl expression system by su er-infection with Ab-MuLV(eco). The secon!d revertant cell line, Rev-2, also retained the Same genome of the v-abl oncogene as the parent line, Ab1-7-2* the tional product of the u-abl oncogene was slight y shorter and no p120gug7ub', the translational product of

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Fig. 3. Detection of u-abl mRNA in transformed and revertant cell lines. Total cellular RNA was extracted by the guanidiudcesium chloride method. Sam les of 10 pg of RNA were separated in 1.4% agarose gel and blottefonto membrane filters. Bands containing u-abl sequences were detected by hybridization with 3zP-labeled pAbl-2 plasmid. Lane M Size markers (indicated in base pairs).

Rev2

N0.7 1

2

3

1

2

3

Rev3 1

2

3

Rev5 1

2

3

P

Abl7-1 1

2

3

Ab17-2 1

1:NRS Fig. 4. Identification of P120 in Ab-MuLV induced transformants and their revertants. Cultures were radiolabeled for 4 hr with 36S-methionine (Amersham, 1,000-1,300 Ciimmol). The cells were lysed and centrifuged, and the cell extracts were immunoprecipitated with normal rat serum (lane l),rat anti-Molony sarcoma virus serum

2

3

Ab17-3 1

2

2:a,MoMSV

3

Ab17-5 1

2

3

M

3 :aGrossMLV

(lane 2), or goat anti-Gross leukemia virus serum (lane 3). Portions of the immunoprecipitated proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis; and the fixed, dried gels were autoradiographed. Track M 14C-labeledmolecular weight markers; numbers are in kilodaltons.

REVERTANTS OF Abl-ONCOGEiiiE TRANSFORMED CELL LINES

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kD

N G N G N G N G N C N G N G

92 -

116

66

-

45

-

Fig. 5 . Detection of autophosphorylation of P12Ww-ab1in transformed and revertant cell lines. Cellular extracts were immunoprecipitated with normal rat serum (N) or SD-rat anti-Gross leukemia virus serum (GI. After the kinase reaction of washed immunoprecipitates with [32P]-gamma-ATP,labeled proteins were separated with 10%SDS-PAGE and detected by autoradiography.

TABLE 3. Suwr-infectionof Ab-MuLVteco)to revertants Dilution rate of virus solution Cells

XI00

x10-1

No. I Rev-2 Rev-3 Rev-5

2,130 1,125 1,720

170'

+

148 119 +2

~

:Transformed foci per plate. Transformed foci were detected; however, exact No. was uncountable.

the v-abl oncogene, or v-ablprotein kinase was detectable, although the ga and env enes of helper virus were expressed norma ly. These Findings suggest that abnormal transcription, induced in some way such as by a mutation or deletion in the v-abl oncogene or regulatory sequence, inhibited normal translation of the v-abl oncogene, resulting in masking of the transformed phenotype. This explanation is supported by the fact that the transformed phenotype was recovered by super-infection with Ab-MuLV(eco)containing the intact v-abl oncogene. The third revertant cell line, Rev-5, expressed

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P120gUg-""to the same extent as the parent transformant, Abl-7-5, and its inte ation of the v-abl oncogene and transcription of v-abl NA were normal. However, it did not show autophosphorylation of the u-abl product, P120g"g"b'. These findings suggest that the P120g"g-ub' in this revertant was functionally impaired by a minor change in the structural gene of the v-abl oncogene such as a point mutation in the active site of the protein kinase reaction. From Southern blot analysis, every revertant was shown to have lost some copies of u-abl oncogene. Such decrease of v-abl oncogene dosage would be possible to affect on cell physiology. In the present study, we found that the relatively high frequency of morphological reversion of the Fisher rat fibroblast line No. 7, transformed by Ab-MuLV was due to segregation and/or inactivation of the v-abl oncogene at multiple sites of gene expression including transcription, translation and inactivation of u-abl protein kinase itself. Analysis of the molecular mechanisms of these reversions in No. 7 cells transformed by Ab-MuLV should be useful in understanding the cellular control system against exogenous genomes in eukaryotic cells. ACKNOWLEDGMENTS One of the a ~ t h o r s(T.O.) thanks Professor Yuji Ohtsuki of Kochi Medical School and Professor Tadaatsu Akagi of Okayama University, School of Medicine, for valuable advice and encouragement. The authors are also grateful to Mrs. Y. Ohshima and Miss M. Ohkochi for preparing this manuscript. LITERATURE CITED

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Baltimore, D., Shields, A,, Otto, G., Goff, S., Besmer, P., Witte, O., and Rosenberg, N. (1980) Structure and expression of the Abelson murine leukemia virus genome and its relationship to normal cell gene. Cold Spring Harbor Symp. Quant. Biol., 44:849454. Chamberlain, J.M. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water-solublefluor, sodium salicylate. Anal. Biochem., 98:132-135. Freeman, A.E., Igel, H.J., and Price, P.J. (1975) In vitro transformation of rat embryo cell; correlations with the known tumorigenic activities of chemicals in rodents. In Vitro, 11f2/:107-116. Glisin, V.R., Crkrenjakov, R., and Byns,,C. (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry, 13:2633. Laemmli, U.K. (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685. Maniatis, T., Fitsch, E.F., and Sambrook,J. (1982)Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 200,201. Reynords, F.H., Jr., Sacks, T.L., Deobagker, D.N., and Stephenson, J.R. (1978) Cells non producing transformed by Abelson murine leukemia virus express a high molecular weight polyprotein containing structural and nonstructural components. Proc. Natl. Acad. Sci. U.S.A., 75:3974-3978. Roberts, J.M., and Axel, R. (1982) Gene amplification and gene correction in somatic cells. Cell, 29:109-119. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis.J. Mol. Biol. 98/31:503517. Witte, O.N., Rosenberg,N., Paskind, H., Shields, A., andBaltimore, D. (1978) Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblast and lymphoid cells. Proc. Natl. Acad. Sci. U.S.A., 75:248&2492.