C-Terminal Amino Acids - Journal of Virology - American Society for ...

Vol. 68, No. 4

JOURNAL OF VIROLOGY, Apr. 1994, p. 2073-2083

0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Transformation of Avian Fibroblasts Overexpressing the c-rel Proto-Oncogene and a Variant of c-rel Lacking 40 C-Terminal Amino Acids JARMILA KRALOVA,t JOHN D. SCHATZLE, WILLIAM BARGMANN, AND HENRY R. BOSE, JR.* Department of Microbiology and the Cell Research Institute, University of Texas at Austin, Austin, Texas 78712-1095 Received 20 September 1993/Accepted 13 December 1993

The v-rel oncogene was derived from the c-rel proto-oncogene, which encodes a transcriptional activator. Expression of v-rel transforms avian hematopoietic cells and fibroblasts. Here we report that overexpression (via a replication-competent retroviral vector) of full-length c-Rel as well as a 40-amino-acid, carboxy-terminal deletion construct of c-Rel (c-RelA) resulted in the morphological transformation of chicken embryo fibroblasts (CEFs). Subcellular localization of Rel polypeptides in these transformed cells as determined by immunofluorescence and immunoprecipitation revealed their presence in both the nucleus and the cytoplasm, with the majority of Rel polypeptides showing cytoplasmic localization. Cytoplasmic localization could be due to interaction with IKB molecules, and in fact, the overexpression of c-Rel or the C-terminal deletion construct of c-Rel resulted in an increase in the levels of mRNA encoding the avian IKB protein pp4O and the avian homolog of the NF-KB protein, p105. However, expression of v-Rel resulted in the induction of pp4O mRNA only. While c-Rel was a weak activator of KB-mediated transcription of a reporter construct in transformed CEFs, v-Rel and c-RelA were transcriptional repressors. However, in spite of these differences, all of these proteins resulted in the transformation of CEFs.

Transformation of avian fibroblasts by v-Rel is associated with changes in morphology, a decrease in actin bundle filaments, anchorage-independent growth in soft agar, extended life span in culture, and the ability to form tumors in vivo (51). In REV-T-transformed lymphoid cells, v-Rel is found in both the cytoplasm and the nucleus, but the majority of the protein is cytoplasmic (25, 26, 50). However, in REVT-infected fibroblasts prior to morphological transformation, v-Rel is found mainly in the nucleus, as determined by immunofluorescence (25, 26, 50). Upon transformation, the majority of v-Rel now becomes principally cytoplasmic as in transformed lymphoid cells (25, 26, 50). In studies in which v-Rel was overexpressed in avian fibroblasts by using a retroviral expression vector, v-Rel was present in both the nucleus and the cytoplasm (51). However, the majority of v-Rel was located in the cytoplasm. Therefore, the presence in both the nucleus and the cytoplasm appears to be a common feature of v-Rel transformation. In fact, removal of the cytoplasmic retention domain of c-Rel allows this mainly cytoplasmic protein to become localized in the nucleus (12, 38). This also enhances the ability of c-Rel to transform avian spleen cells, indicating that nuclear localization may be a contributing factor in transformation. In this report, we demonstrate that overexpression of v-Rel, full-length c-Rel, and a variant of c-Rel lacking 40 C-terminal amino acids (c-RelA) resulted in the transformation of chicken embryo fibroblasts (CEFs). In addition, injection of newborn chickens with these transformed fibroblasts produced fibrosarcomas. In cells overexpressing these proteins, Rel polypeptides were found in both the nucleus and the cytoplasm. Overexpression of either full-length c-Rel or the truncated form of c-Rel as well as v-Rel resulted in an increase in the steady-state levels of mRNA encoding avian IKBo, pp4O. Overexpression of c-Rel and the carboxy-terminal deletion construct of c-Rel

Avian reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic retrovirus that transforms cells of myeloid and lymphoid origin both in vivo and in vitro (reviewed in references 6, 24, and 57). REV-T encodes a 59-kDa protein (v-Rel) which is responsible for this transformation (25, 29). v-rel is a transforming variant of the avian cellular protooncogene, c-rel. v-Rel is missing two N-terminal and 118 C-terminal amino acids present in c-Rel (14, 69, 77). The deletion of the C-terminal amino acids of c-Rel results in the removal of both a transcriptional activation domain and a cytoplasmic retention domain (12, 18, 38). In addition, v-Rel contains multiple amino acid substitutions and three small in-frame deletions which may also contribute to its oncogenic potential (77). Both c-Rel and v-Rel exhibit extensive homology to known transcription factors, including the NF-KB transcription complex (p50 and p65), the Drosophila melanogaster morphogen dorsal, and other Rel-related factors (RelB, p49, and x-Rel) (2, 3, 23, 40, 44, 45, 62, 65, 70). In addition, both c-Rel and v-Rel have been shown to dimerize with NF-KB subunits (3, 22, 30, 43, 56). While c-Rel functions as a transcriptional activator of promoters containing KB binding sites, v-Rel is a dominant negative repressor of these same promoters (3, 9, 35, 48, 59). Nontransforming mutants of v-Rel are unable to repress KB-dependent transcription (3, 53). In addition, removal of the C-terminal transcriptional activation domain of c-Rel results in a variant of c-Rel which is now able to transform avian spleen cells (38). These studies indicate that there may be a correlation between repression of KB-dependent transcription by Rel proteins and their ability to transform cells. * Corresponding author. Phone: (512) 471-5525. Fax: (512) 4717088. t On sabbatical from the Institute of Molecular Genetics, Czech Academy of Sciences, 14220 Prague 4, Czech Republic.

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KRALOVA ET AL.

resulted in an induction of mRNA encoding avian p105, while

overexpression of v-Rel did not.

MATERIALS AND METHODS Cells and tissue culture. CEFs were prepared from 11-dayold SPAFAS embryos (Norwich, Conn.). CEFs were maintained in culture in Eagle's minimal essential medium (JRH Biosciences) supplemented with 6% fetal calf serum, penicillin (100 U/ml), and streptomycin (50 ,ug/ml). Murine F9 embryonal carcinoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Differentiation of F9 cells was accomplished by incubation in 5 x 10-' M retinoic acid for 3 days. Plasmid constructs. The avian c-rel cDNA (3,727 bp) cloned into the pBluescript vector (12) was used to construct expression vectors by using the replication-competent avian retroviral vector, RCAS (34). First, an EcoRV-NaeI fragment containing the coding sequences of c-rel but lacking the 3' untranslated region was subcloned into the SmaI site of the Clal2 adaptor plasmid (34). A ClaI fragment containing the c-rel cDNA was then subcloned from the Clal2 plasmid into the ClaI site downstream of the 3' splice acceptor site of the RCAS vector to create the RCAS-c-Rel construct. The carboxy-terminal deletion construct of c-Rel (c-RelA) was generated by PCR amplification. The primers used for PCR were constructed to introduce an in-frame stop codon and a unique EagI restriction site into the c-rel gene. A TAA stop codon was introduced at nucleotide 1738, which should result in the synthesis of a c-Rel protein lacking 40 C-terminal amino acids. The resulting PCR product was digested with BamHI and EagI, and this fragment (829 bp) was used to replace the corresponding fragment in the c-rel cDNA cloned into pBluescript. Then a BamHI-XbaI fragment (774 bp) from the wild-type c-rel cDNA was used to replace the corresponding fragment in the mutated c-rel cDNA. This resulted in the replacement of only 55 bp of the original wild-type c-rel cDNA with the mutated PCR fragment. This 55-bp region was completely sequenced to ensure that no other mutations besides the stop codon were introduced into this region by PCR. The mutated c-rel (c-relA) cDNA was subcloned into the Clal2 adaptor plasmid by digesting the c-relA cDNA with EagI, which was then blunt ended by treatment with Klenow enzyme and digested with EcoRV (site located in the polylinker of pBluescript). This EcoRV-EagI fragment was subcloned into the SmaI site of pClal2. A ClaI fragment containing the c-relA cDNA was then subcloned from the Clal2 vector into the ClaI site of the RCAS vector. DNA transfections and chloramphenicol acetyltransferase (CAT) assays. Plasmid DNA was introduced into CEFs by the calcium phosphate precipitation method (28). Briefly, cells were plated at a density of 1.5 x 106 cells per 60-mm-diameter plate 1 day prior to transfection. Cells were then washed in phosphate-buffered saline (PBS), and 1 ml of DNA precipitate was added to the cells for 1 h at 37°C followed by the addition of 5 ml of Eagle's minimal essential medium containing 100 FiM chloroquine. Cells were then incubated for 3 h at 37°C, the medium was aspirated, and cells were treated with 15% glycerol for 3 min at room temperature. This was followed by a complete medium change, and cells were then maintained in culture and passaged at 3-day intervals.

Transfection of the transient reporter constructs (KB)6TKCAT or (KBM)6TK-CAT into CEFs already overexpressing Rel polypeptides was accomplished as follows. Cells (2.5 x 106/100-mm-diameter plate) were transfected with 3 ,ug of reporter construct, and pBluescript was added to adjust the final amount of DNA to 18 ,g. The DNA precipitate was left

J. VIROL.

in contact with the cells for 4 to 12 h, cells were then incubated with 15% glycerol for 2 min, and the medium was exchanged. Cells were incubated for 30 h and then harvested. Cell extracts were prepared by three cycles of freeze-thawing, and extracts were normalized according to total protein levels (8). CAT activity was determined by incubating cell extracts with ['4C]chloramphenicol and acetyl coenzyme A for 1 h as previously described (27). CAT activity was expressed as percent conversion of the '4C substrate into acetylated products, as determined by scintillation counting of excised spots. F9 cells were differentiated by the addition of 5 x 10-7 M retinoic acid 3 days prior to transfection. F9 cells were cotransfected with the reporter constructs mentioned above along with the c-rel, c-relA, and v-rel cDNAs cloned into the RCAS expression vector. Transfections were repeated three to five times with at least two different plasmid preparations. All plasmids were purified by using CsCl gradients or Qiagen columns. Colony formation in soft agar. Transfected CEFs (105 cells) passaged for 3 weeks following transfection were plated in 5 ml of Dulbecco's modified Eagle's medium containing 15% fetal calf serum, 2% chicken serum, and 0.35% Noble agar per 60-mm-diameter dish containing a bottom layer of 0.7% agar. Cells were refed with additional soft agar medium following 1and 2-week periods. Plates were scored for the development of colonies 2 to 3 weeks following plating. Western immunoblot analysis. Cell lysates of transfected CEFs were prepared as previously described (46); 50 pLg of total cellular protein was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis followed by electrophoretic transfer to nitrocellulose membranes (80). Western blot analysis was performed as previously described, using rabbit polyclonal antiserum to c-Rel (20), pp40 (15), or v-Rel (76). Secondary antibody was alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.). Immunofluorescence. Transfected CEFs were grown on glass coverslips, washed several times with PBS, and then fixed with 2% paraformaldehyde in PBS for 20 min at room temperature. Following two PBS washes, cells were permeabilized for 15 min with 0.1% Triton X-100 in PBS, washed once with PBS, and incubated for two 10-min intervals with 3% bovine serum albumin (BSA) in PBS. Fixed cells were then incubated for 1 h at 37°C with the anti-c-Rel monoclonal antibody 1OH, derived to the JZ49 fusion protein (20), at a 1:100 dilution. Coverslips were then washed four times in PBS with BSA for 15 min each time and then incubated for 1 h at 37°C with a 1:50 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (Kirkegaard & Perry). Coverslips were washed again in PBS and mounted on slides, using PBS-buffered glycerol. Subcellular fractionation and immunoprecipitations. CEFs were starved for 90 min in methionine-cysteine-free medium containing 5% fetal calf serum. Cells were then labeled with 175 ,uCi of [35S]methionine-cysteine label mix (NEN Pro Lab mix) per ml, using 3.5 ml/100-mm-diameter plate for 4.5 h at 37°C. Cells were harvested after three washes with cold PBS by scraping with a rubber policeman into lysis buffer containing protease inhibitors (15). Lysates were spun in a microcentrifuge, and supernatants were counted in a liquid scintillation counter. Immunoprecipitations were done on equivalent counts per minute and were performed as previously described (15). Rabbit polyclonal antiserum specific for p105 was generated by the injection of a fusion protein consisting of 175 N-terminal amino acids of p105 fused to the bacterial TrpE protein. This antiserum showed reactivity to only p105 in

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TRANSFORMATION OF AVIAN FIBROBLASTS BY c-rel

Western blot analysis and did not react with v-Rel or c-Rel (data not shown). For subcellular fractionations, labeled cells were harvested and resuspended in hypotonic lysis buffer (20 mM Tris [pH 7.8], 20 mM KCl, 1 mM MgCl2, protease inhibitor cocktail) and allowed to swell for 15 min on ice. Cells were then subjected to Dounce homogenization, and lysates were then centrifuged at 2,000 rpm for 2 min in a Beckman TJ-6 centrifuge. Soluble lysates (containing the cytosolic proteins) were spun in a microcentrifuge, and the supernatants were used for immunoprecipitation as described above. The crude nuclear pellet was gently resuspended in hypotonic lysis buffer, and nuclei from 10 unlabeled cells were added as a carrier for nuclear fractionation. A 40% (wt/vol) sucrose solution in hypotonic lysis buffer was added to these crude nuclei in a 3:1 ratio. These samples were then loaded onto a 66% sucrose cushion and centrifuged at 28,000 rpm for 45 min in a Beckman SW50.1 rotor. The nuclear pellet was then resuspended in 1 x immunoprecipitation buffer and subjected to immunoprecipitation as previously described (15). Northern (RNA) blot analysis. Total cellular RNA was isolated by the lithium chloride-urea procedure (1). Twenty micrograms of total RNA was loaded per lane and fractionated by formaldehyde agarose gel electrophoresis (64). This was followed by capillary transfer to nylon membranes (Nytran; Schleicher & Schuell), which were then stained with methylene blue to confirm equal loading and transfer of RNA (66). Filters were then hybridized at 65°C with one of the following radiolabeled probes: pp40 cDNA (16), p105 cDNA (10), and actin cDNA fragment (20). Hybridizations and washes were performed as previously described (64). Autoradiographs and photographs of stained membranes were scanned by a Bio-Rad laser densitometer. RESULTS Overexpression of c-Rel and a carboxy-terminal deletion construct of c-Rel results in the transformation of CEFs. The avian c-Rel protein has been shown to contain multiple transactivation domains (18, 38). To investigate the importance of transactivation domain II (12, 38) in the biological activity of c-Rel in CEFs, we generated a carboxy-terminal deletion construct of c-Rel (c-RelIA) that also lacks the 3' noncoding sequences of the c-rel cDNA. This was accomplished by introducing a stop codon (TAA) following nucleotide 1738 of the c-rel cDNA (12), which should result in the synthesis of a 558-amino-acid polypeptide that is 40 amino acids shorter than the full-length c-Rel protein. The c-rel cDNA (lacking the 3' noncoding sequences) and the Cterminal deletion construct (c-RelA) were cloned into the replication-competent avian retroviral vector RCAS (all constructs were in the A envelope subgroup unless otherwise indicated). These constructs along with a construct containing v-rel cloned into the RCAS vector (34) were used to transfect CEFs to examine the effects of overexpression of these various proteins. Transfections were routinely performed on CEFs following the second or third passage, and at least two more passages were required following transfection to detect the expression of the transfected genes. Figure 1A is a Western blot of representative transfected CEFs and shows that we were able to obtain cell cultures overexpressing c-Rel (p68), c-RelA (p64), and v-Rel (p59) (lanes 2 to 4). Overexpression of c-RelA did not affect the steady-state levels of endogenous c-Rel (compare lanes 1 and 3). The additional 64-kDa immunoreactive protein seen in lanes 1 and 2 corresponds to the previously described Rel-related protein found in CEFs (20).

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Even though we observed variations in the levels of expression of the transfected genes, the results from 10 independent experiments revealed that, approximately 5 to 10 passages following transfection of CEFs with the v-Rel construct, cells began to lose the characteristic shape of fibroblasts and began to assume a more cuboidal and enlarged shape (Fig. 1E). This morphological change was also observed following 8 to 15 passages for cells overexpressing c-RelA (Fig. ID) and following 12 to 20 passages for cells overexpressing c-Rel (Fig. 1C). However, transformation of CEFs transfected with RCAS-cRel was obtained only when very high levels (5- to 10-fold overexpression compared with endogenous c-Rel) of c-Rel were expressed (representing approximately 50% of all transfections). In contrast, cells transfected with the RCAS vector alone exhibited no change in morphology (Fig. 1B). In addition, cells cotransfected with a c-Rel antisense expression vector along with the RCAS-c-RelA vector exhibited no

morphological changes (data not shown).

To further characterize the transformation of CEFs by the overexpression of Rel proteins, we measured the growth rate

of these cells. The v-Rel-transfected cultures showed increased

growth rates at 7 to 10 generations posttransfection. Increased growth rates for cells overexpressing c-RelA (p64) and c-Rel (p68) were seen at 10 to 15 and 15 to 20 generations posttransfection, respectively (data not shown). Increased growth rates

correlated with an increase in the number of cells overexpressing Rel polypeptides and also correlated with morphological changes of these cultures (data not shown). Cells transfected with RCAS-c-RelA or RCAS-c-Rel showed an increased growth rate when greater than 90% of the cells were positive for overexpression. In contrast, we did not observe an increase in the growth rate of cells transfected with the RCAS vector alone. Finally, we observed an extended culture life span for CEFs overexpressing c-Rel and c-RelA (Table 1), as previously reported for cells overexpressing v-Rel (51). It has been reported that the morphological change associated with the transformation of CEFs correlates with a decrease in the number of actin filament bundles (51, 60). The morphological changes observed in cells overexpressing v-Rel, c-Rel, or c-RelA also correlated with a decrease in the steady-state levels of actin mRNA compared to CEFs transfected with the RCAS vector alone (see Fig. 5D). These results suggest that morphological transformation of CEFs by the overexpression of these various Rel polypeptides results in a decrease in actin mRNA, which should result in a decrease in actin filament bundles and could contribute to the morphological changes observed. To further characterize the transformation potential of c-Rel and c-RelA on CEFs, transfected cells were tested for the ability to form colonies in soft agar. Figure 2A to C shows that CEFs transfected with the c-RelA (Fig. 2C) or c-Rel (Fig. 2B) expression construct were able to form colonies in soft agar at low efficiency (0.04 and 0.01%, respectively), whereas CEFs transfected with the RCAS vector alone (Fig. 2A) could not. The efficiency of colony formation for CEFs transfected with an RCAS-v-Rel expression vector has been reported to be 0.05% (52). However, cells overexpressing c-Rel formed fewer and smaller colonies than those formed by CEFs overexpressing c-RelA (Table 1). These results were consistently observed in several independent experiments. The observations described above indicate that overexpression of c-Rel, c-RelA, or v-Rel in CEFs can result in the morphological transformation of these cells. However, the crucial test of transformation is the ability of these cells to be tumorigenic in vivo. Therefore, we tested the in vivo tumorigenic potential of CEFs overexpressing c-Rel, c-RelA, or v-Rel

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J. VIROL.

KRALOVA ET AL.

A

cc-te

Anti-c-rel

Anti-v-rel

M. 1

2

3

4

B

F

FIG. 1. Overexpression of c-Rel (p68), c-RelA (p64), and v-Rel (p59) in CEFs is associated with morphological changes. (A) Cells transfected with the RCAS (lane 1), RCAS-c-Rel (lane 2), RCAS-c-RelA (lane 3), or RCAS-v-Rel (lane 4) vector were harvested, and 50 p.g of total protein was subjected to SDS-PAGE followed by Western blot analysis as described in Materials and Methods. Lanes 1 to 3 were incubated with primary antibodies to c-Rel, and lane 4 was incubated with primary antibodies to v-Rel. (B to E) Transfected cells were passaged in vitro until morphological changes were observed and then photographed at a magnification of x 200. CEFs transfected with RCAS (B), RCAS-c-Rel (C), RCAS-c-RelA (D), or RCAS-v-Rel (E) are represented.

by injecting these cells into the wing webs of 1-day-old SPAFAS chicks. This resulted in a high percentage of fibrosarcoma development at the site of injection (Fig. 3D and E; Table 1). However, no fibrosarcomas were detected when CEFs transfected with the RCAS vector alone were injected into chicks. Dissection of chickens that died as a result of the injection of CEFs overexpressing c-Rel, c-RelA, or v-Rel revealed the presence of enlarged spleens and characteristic tumor foci on the liver (data not shown). Tumors formed by the injection of cells overexpressing v-Rel developed quickly and were often fatal within 3 weeks following injection. Interestingly, tumors resulting from the injection of CEFs overexpressing c-RelA fell into one of two categories, depending on the envelope subgroup of the retroviral expression vector. CEFs transfected with the c-RelA RCAS(D) vector showed faster tumor development (similar to that seen with

v-Rel) and resulted in the death of injected chickens. However, CEFs transfected with the c-RelA RCAS(A) vector showed tumor development in injected chicks occasionally more than 1 month postinjection. The difference in the kinetics of tumor development is presumed to reflect the relative abundance of env receptors for each subgroup. In addition, CEFs overexpressing c-Rel only occasionally produced tumors when injected into chicks, and this tumorigenic potential correlated with the in vitro transformed phenotype of these cells. Table 1 summarizes observations regarding the transforming properties of CEFs overexpressing c-Rel, c-RelA, or v-Rel. From these observations, we conclude that overexpression of c-RelA is nearly as oncogenic as overexpression of v-Rel. Overexpression of c-Rel was not as oncogenic as overexpression of c-RelA or v-Rel. However, in CEFs, in which c-Rel was highly overexpressed in the majority of cells (as determined by

TABLE 1. Summary of transformation parameters

Construct

Morphological changes

Soft agar colony (CFU/105 cells formation plated)

Increased growth rate

Life span (generations)

Sarcoma formation (no. of tumors/no.

in Reduction actin mRNA

4/14 15/19 4/6

+ + +

of animals injected)

RCAS c-Rel c-RelA v-Rel a

ND, not determined.

+ + +

± (10) + (40)

NDa

+ + +

23-30 30-45 35-50 45-60

A.

B

C

0'jt0.;7ftrw

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TRANSFORMATION OF AVIAN FIBROBLASTS BY c-rel

VOL. 68, 1994

D

A c-el

RCAS

B RCAS-c-rel

-ow.

..

c-relA

C

RCAS-c-reIA

i

IN

C'

N

C'

N

C'

N

E D

S0 tED.;t4t

w

i*>,,:',.

S ^^