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. 1998 May;18(5):2997-3009.
doi: 10.1128/mcb.18.5.2997.

AP-1 Factors Play an Important Role in Transformation Induced by the V-Rel Oncogene

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Free PMC article

AP-1 Factors Play an Important Role in Transformation Induced by the V-Rel Oncogene

J Kralova et al. Mol Cell Biol. .
Free PMC article

Abstract

v-rel is the oncogenic member of the Rel/NF-kappaB family of transcription factors. The mechanism by which v-Rel induces transformation of avian lymphoid cells and fibroblasts is not precisely known. However, most models propose that v-rel disrupts the normal transcriptional regulatory network. In this study we evaluated the role of AP-1 family members in v-Rel-mediated transformation. The overexpression of v-Rel, c-Rel, and c-Rel delta resulted in a prolonged elevation of c-fos and c-jun expression and in a sustained repression of fra-2 at both the mRNA and protein levels in fibroblasts and lymphoid cells. Moreover, the transforming abilities of these Rel proteins correlated with their ability to alter the expression of these AP-1 factors. v-Rel exhibited the most pronounced effect, whereas c-Rel, with poor transforming ability, elicited only moderate changes in AP-1 levels. Furthermore, c-Rel delta, which exhibits enhanced transforming potential relative to c-Rel, induced intermediate changes in AP-1 expression. To directly evaluate the role of AP-1 family members in the v-Rel transformation process, a supjun-1 transdominant mutant was used. The supjun-1 mutant functions as a general inhibitor of AP-1 activity by inhibiting AP-1-mediated transactivation and by reducing AP-1 DNA-binding activity. Coinfection or sequential infection of fibroblasts or lymphoid cells with viruses carrying rel oncogenes and supjun-1 resulted in a reduction of the transformation efficiency of the Rel proteins. The expression of supjun-1 inhibited the ability of v-Rel transformed lymphoid cells and fibroblasts to form colonies in soft agar by over 70%. Furthermore, the expression of supjun-1 strongly interfered with the ability of v-Rel to morphologically transform avian fibroblasts. This is the first report showing that v-Rel might execute its oncogenic potential through modulating the activity of early response genes.

Figures

FIG. 1
FIG. 1
Expression levels of mRNAs encoding AP-1 components in Rel-overexpressing cells. (A) Expression of c-jun and junD mRNA in CEF cultures infected with viruses carrying no insert (DS) or the oncogene c-relΔ (CΔ) or v-rel (V). A 15-μg sample of each RNA was used for Northern blot hybridization, first with radiolabeled c-jun cDNA and then with a human GAPDH probe to demonstrate the RNA loading (left two panels). For junD mRNA analysis, 20 μg of total RNA was used and the membrane was hybridized with junD cDNA followed by rehybridization with GAPDH (right two panels). (B) Serum stimulation of c-fos and fra-2 mRNA. Three different cell pools of DS3-, c-relΔ-, and v-rel-infected CEF cultures were grown in serum (exponentially growing cells [E]), or were serum starved for 18 h in the presence of 0.2% calf serum (quiescent cells [G0]) and then stimulated by the addition of 10% calf serum for 90 min prior to RNA extraction (serum-stimulated cells [G1]). Total RNA (20 μg) isolated from these various cultures was sequentially analyzed by Northern blot hybridization with radiolabeled chicken c-fos, fra-2, and human GAPDH DNA probes. (C) Expression levels of endogenous c-jun and fra-2 mRNA in infected DT95 lymphoid cells. DT95 cells were infected with REV-C (C) or REV-T (T) in the presence of the CSV helper virus. Three weeks after infection, the expression of these oncoproteins was confirmed by Western blot analysis (see Fig. 2B) and total RNA (20 μg) was subjected to Northern blot analyses with c-jun and fra-2 DNA probes as described above. (D) Time course of c-fos mRNA induction in CEF cultures following infection with a retrovirus expressing v-rel. Total RNA (20 μg per lane) was extracted at the time points (days) indicated and sequentially analyzed by Northern blot hybridization with radiolabeled c-fos and then GAPDH probes.
FIG. 2
FIG. 2
Expression of endogenous c-Jun, Fra-2, and c-Fos in cells overexpressing Rel proteins. (A) Proteins in whole-cell lysates from control and c-relΔ- or v-rel-expressing CEF cultures (each analysis contained the equivalent of 2 × 105 cells) were resolved on SDS–10% polyacrylamide gel and then subjected to immunoblotting with the enhanced chemiluminescence Western blotting detection system. Membranes were cut into two parts; the upper portion was incubated with anti-v-Rel (α v-Rel) serum to detect p59v-rel or anti-c-Rel serum (α c-Rel) to detect p64c-relΔ, and the lower portion was incubated with anti-c-Jun (α c-Jun) antiserum to detect c-Jun (40 kDa). The expression of these proteins was monitored at 10 days postinfection. (B) Proteins in whole-cell lysates corresponding to 2 × 105 cells per lane from DT95 infected with REV-T (v-rel) or REV-C (c-rel) in the presence of the CSV helper virus were analyzed by Western blot analysis as described above for CEF cultures. (C) Exponentially growing (E) and serum-stimulated (G1) CEF cultures (expressing v-rel [V] or DS3 [DS]) were 35S labeled for 60 min, and proteins in nuclear extracts were immunoprecipitated with anti-Fra-2 serum (lanes 1 to 4). Then the supernatant fluids from these Fra-2 precipitations were subjected to a second precipitation with anti-c-Jun serum (lanes 5 to 8) to detect coprecipitated c-Fos. Proteins in immunoprecipitates were resolved by SDS-PAGE (10% polyacrylamide) and visualized by autoradiography after exposure of the X-ray film for 3 weeks.
FIG. 3
FIG. 3
Effect of supjun-1 expression on the level of endogenous c-jun. (A) Level of the c-jun transcript in CEF cultures infected with DS3 and supjun-1 viruses (top panel). CEF cultures were harvested 10 days after infection, and total RNA was extracted and subjected to Northern blot analyses (20 μg per lane) with a radiolabeled c-jun cDNA probe. RNA loading was demonstrated by showing 28S rRNA (bottom panel). (B) Nuclear extracts (50 μg) from infected CEF cultures were analyzed on a discontinuous 4, 10, and 18% step polyacrylamide gel as described in Materials and Methods and then subjected to immunoblotting. After blotting, the membranes were cut into two parts. The top half was incubated with anti-c-Jun serum to detect endogenous c-Jun (40 kDa), and the bottom half was incubated with a panspecific Jun antiserum to detect the human supJun protein. (C) Nuclear extracts (50 μg) from v-Rel-transformed lymphoid cells C4-1 and 160/2 were isolated, and protein expression was analyzed 10 days after superinfection with DS3 or supjun-1 viruses as described for panel B.
FIG. 4
FIG. 4
(A) Inhibition of c-Jun-induced transactivation of collagenase promoter by supjun-1. Transactivation was assessed by measuring CAT activity in extracts of CEF cultures cotransfected with or without supjun-1 and the −73/+63coll CAT reporter gene (lanes 1 to 6, solid bars) or −60/+63coll CAT with the AP-1 site deleted (lanes 7 and 8, open bars) 24 h posttransfection (34). (B) Inhibition of v-Rel-induced transactivation by supjun-1. CEF cultures were cotransfected with expression vectors for the genes indicated, along with a luciferase reporter construct with or without multiple AP-1 binding sites cloned in front of the SV40 promoter, respectively (lanes 1 to 7, solid bars; lanes 8 to 12, open bars). Luciferase activity for equal amounts of protein was determined as described in Materials and Methods 36 h after transfection (65). The fold activation was determined by dividing the actual chloramphenicol or luciferase activity obtained in the presence of c-Jun, c-Fos, v-Rel, and supJun expression vectors by the activity of the reporter when the cells were cotransfected with an “empty” expression vector, pRc/RSV (defined as baseline activity 1.0). The mean results from three to five experiments are shown. Standard errors were determined; however, in some cases they are not visible in the given activation scale for the luciferase assays.
FIG. 5
FIG. 5
(A) Activation of the collagenase promoter in Rel-transformed CEF cultures. Transactivation was assayed by measuring CAT activity in extracts of v-Rel- or c-RelΔ-transformed CEF cultures transfected with the −73/+63coll CAT reporter gene (lanes 1 to 4, solid bars) or −60/+63coll CAT with the AP-1 sites deleted (lanes 5 to 8, empty bars) 24 h posttransfection. Lanes: 1 and 5, uninfected CEF cultures; 2 and 6, DS3-infected CEF cultures; 3 and 7, c-RelΔ-transformed CEF cultures; 4 and 8, v-Rel-transformed CEF cultures. (B) Activation of (AP-1)5SV40-luc in Rel-transformed CEF cultures. Lanes: 1 and 5, uninfected CEF cultures; 2 and 6, DS3-infected CEF cultures; 3 and 7, c-RelΔ-transformed CEF cultures; 4 and 8, v-Rel-transformed CEF cultures. Luciferase activity for equal amounts of protein was determined as described in Materials and Methods 36 h after transfection (65). Fold activation was determined by dividing the actual chloramphenicol or luciferase activity obtained from v-Rel- or c-RelΔ-transformed CEF cultures by the activity of the reporter from normal CEF cultures or those infected with an “empty” expression vector, DS3 (defined as baseline activity 1.0). The mean results from two experiments with standard errors are shown.
FIG. 6
FIG. 6
(A) DNA-binding activity of in vitro-translated c-Jun, c-Fos, and supJun. Proteins were synthesized separately by using a reticulocyte lysate-based in vitro transcription-translation system and then mixed and incubated at 37°C for 45 min to allow dimerization. The DNA-binding activity was determined by an EMSA with 1 ng of a 32P-labeled oligonucleotide containing the AP-1-binding site (FSE2) and 4.5 μl of reticulocyte lysate mixture in the presence (+) (lanes 3, 5, and 11) or absence (−) (lanes 2, 4, and 6 to 10) of a 100-fold molar excess of unlabeled oligonucleotide. The positions of the shifted c-Jun-, c-Fos-, and supJun homo- and heterodimer-containing complexes are indicated by the numbers 1 to 5 (1, c-Jun/c-Jun; 2, c-Jun/c-Fos; 3, supJun/c-Jun; 4, supJun/c-Fos; 5, supJun/supJun). The complex labeled e is due to the reticulocyte lysate since it was seen when unprogrammed lysate was mixed with the oligonucleotide (lane 1). (B) Complex formation between oligonucleotides containing consensus AP-1 or mutant AP-1 sequences and nuclear proteins from CEF cultures. Nuclear extracts (5 μg) from CEF cultures infected with viruses carrying no insert (DS3) (lanes 1 and 4), v-rel (lanes 2 and 5), or c-relΔ (lane 3 and 6) were assayed 10 days after infection for AP-1-binding activity with 0.2 ng of a 32P-labeled AP-1 oligonucleotide (lanes 1 to 3) or mutant AP-1 oligonucleotide (lanes 4 to 6) (Santa Cruz). Bands A and B represent AP-1 DNA-binding activity in DS3- and Rel-infected CEF cultures, respectively. (C) Endogenous AP-1 DNA-binding activity in nuclear extracts from infected CEF cultures and lymphoid cells. Nuclear extracts (5 μg) from CEF cultures infected with viruses carrying no insert (DS3) (lanes 1 and 14), v-rel (lanes 2, 6, 8, and 15), c-relΔ (lane 3), or supjun-1 (lanes 4, 7, 16, and 18) or coinfected with v-rel and supjun-1 (v + sj) (lanes 5 and 17) were assayed 10 days after infection for AP-1-binding activity with 0.2 ng of a 32P-labeled AP-1 oligonucleotide (Santa Cruz). Lanes 1 to 8 and 14 to 18 show binding activity in nuclear extracts isolated from CEF cultures, whereas lanes 9 to 13 show activity originating from v-Rel-transformed C4-1 lymphoid cells superinfected with DS3 (lanes 9 and 12) or supjun-1 (lanes 10, 11, and 13) viruses (2.5 μg per lane). The specificity of AP-1 binding was determined by competition assays with a 100-fold molar excess of unlabeled oligonucleotide (+) or mutant oligonucleotide (m). The top bands, A and B (lane 8), are most probably formed by dimers of the Fos/Jun family. The lower bands, C and D, were detected in nuclear extracts from supJun-expressing cells (lanes 4, 5, and 10) and represent supJun heterodimers with Fos/Jun proteins and supJun homodimers, respectively.
FIG. 7
FIG. 7
Cellular morphology of sequentially infected CEF cultures. CEF cultures were infected with the virus from subgroup A first and superinfected 4 days later with the virus from subgroup B to deliver the second gene. (A) CEF cultures infected with the virus carrying no insert, DS3. (B and C) CEF cultures infected first with c-relΔ followed by DS3 (B) or supjun-1 (C). (F and G) CEF cultures infected first with v-rel followed by DS3 (F) or supjun-1 (G). When the order of introduction of the rel genes and supjun-1 or DS3 was reversed, the inhibitory effect of supJun expression on the Rel-induced transformation was not substantially changed: DS3 followed by v-rel resulted in a transformed phenotype (E), whereas infection of CEF cultures with the supjun-1 rendered them resistant to subsequent transformation by v-Rel (H) or c-RelΔ (D). Infected cells were passaged in vitro for 10 days after the second superinfection and then photographed at a magnification of ×200.

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