ERK and JNK activation is essential for oncogenic transformation by v-Rel.

v-Rel is the acutely oncogenic member of the NF-κB family of transcription factors. Infection with retroviruses expressing v-Rel rapidly induces fatal lymphomas in birds and transforms primary lymphocytes and fibroblasts in vitro. We have previously shown that AP-1 transcriptional activity contributes to v-Rel-mediated transformation. Although v-Rel increases the expression of these factors, their activity may also be induced through phosphorylation by the mitogen-activated protein kinases (MAPKs). The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways. This induction is critical for the v-Rel-transformed phenotype, as suppression of MAPK activity with chemical inhibitors or small interfering RNA severely impairs colony formation of v-Rel-transformed lymphoid cell lines. However, signaling must be maintained within an optimal range in these cells, as strong additional activation of either pathway beyond the levels induced by v-Rel through the expression of constitutively active MAPK proteins attenuates the transformed phenotype. MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified. We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

Cell transformation by v-Rel reveals distinct roles of AP-1 family members in Rel/NF-kappaB oncogenesis.

Cell transformation by the v-rel oncogene is mediated by the aberrant expression of genes that are normally tightly regulated by other Rel/NF-kappaB family members. Although a number of genes inappropriately activated or suppressed by v-Rel have been identified, their contributions to the v-Rel transformation process have been poorly characterized. Here, we examine the role of individual AP-1 proteins in v-Rel-mediated transformation. v-Rel-transformed cells exhibit elevated RNA and protein expression of c-Fos, c-Jun and ATF2 and sustained repression of Fra-2. c-Fos and c-Jun are essential in both the initiation and maintenance of v-Rel-mediated transformation, whereas Fra-2 is dispensable. By employing a c-Jun dimerization mutant, we further identified Fos/Jun heterodimers as major contributors to the v-Rel transformation process. The inability of c-Rel to induce the expression of c-Fos and c-Jun contributes to its weaker oncogenic potential relative to v-Rel. Our studies also demonstrate that v-Rel may induce AP-1 members by directly upregulating gene expression (c-fos and ATF2) and by activating pathways that stimulate AP-1 activity. Although elevated expression of ATF2 is also required for v-Rel-mediated transformation, its ectopic overexpression is inhibitory. Investigating the mode of ATF2 regulation revealed a positive feedback mechanism whereby ATF2 induces p38 MAPK phosphorylation to further induce its own activity. In addition, these studies identified Ha-Ras as an effector of v-Rel-mediated transformation and reveal a novel role for ATF2 in the inhibition of the Ras-Raf-MEK-ERK signaling pathway. Overall, these studies reveal distinct and complex roles of AP-1 proteins in Rel/NF-kappaB oncogenesis.

The activation of TC10, a Rho small GTPase, contributes to v-Rel-mediated transformation.

v-Rel is the oncogenic member of the Rel/NF-kappaB family of transcription factors and transforms hematopoietic cells and fibroblasts. Differential display was employed to identify target genes that exhibit altered expression in v-Rel transformed cells. One of the cDNAs identified encodes the chicken ortholog of TC10, a member of the Rho small GTPase family. The expression of TC10 was increased in v-Rel-transformed chicken embryonic fibroblasts (CEFs) 3 to 6-fold relative to control cells at both the RNA and protein levels. An elevated level of active, GTP-bound TC10 was also detected in v-Rel-transformed cells relative to control cells. Expression of a dominant-negative TC10 mutant (TC10T32N) decreased the colony formation potential of v-Rel-transformed cells. Furthermore, overexpression of wild-type TC10 or a gain-of-function mutant (TC10Q76L) greatly enhanced the ability of v-Rel transformed CEFs to form colonies in soft agar. In addition to enhance the transformation potential of v-Rel, the overexpression of wild-type TC10 or the gain-of-function mutant alone enhanced the saturation density of CEFs and was sufficient for their anchorage-independent growth in vitro. These results indicate that elevated TC10 activity contributes to v-Rel-mediated transformation of CEFs and demonstrate for the first time that a Rho factor alone is capable of inducing the in vitro transformation of primary cells.

The suppression of SH3BGRL is important for v-Rel-mediated transformation.

The v-rel oncogene is the most efficient transforming member of the Rel/NF-kappaB family of transcription factors. v-Rel induces avian and mammalian lymphoid cell tumors and transforms chicken embryo fibroblasts in culture by the aberrant regulation of genes under the control of Rel/NF-kappaB proteins. Here we report that the expression of SH3BGRL, a member of the SH3BGR (SH3 domain-binding glutamic acid-rich) family of proteins, is downregulated in v-Rel-expressing fibroblasts, lymphoid cells, and splenic tumor cells. Chromatin immunoprecipitation experiments demonstrated that v-Rel binds to the sh3bgrl promoter in transformed cells. Coexpression of SH3BGRL with v-Rel in primary splenic lymphocytes reduced the number of colonies formed by 76%. Mutations in the predicted SH3-binding domain of SH3BGRL abolished the suppressive effect on v-Rel transformation and resulted in colony numbers comparable to those formed by v-Rel alone. However, mutations in the predicted EVH1-binding domain of SH3BGRL only had a modest effect on suppression of v-Rel transformation. This study provides the first example of a gene that is downregulated in v-Rel-expressing cells that also plays a role in v-Rel transformation.

The chicken RelB transcription factor has transactivation sequences and a tissue-specific expression pattern that are distinct from mammalian RelB.

Rel/NF-kappaB proteins are eukaryotic transcription factors that control the expression of genes involved in a large variety of cellular processes. Rel proteins share a highly conserved DNA-binding/dimerization domain called the Rel Homology (RH) domain. We have constructed and characterized a composite cDNA encoding most of the chicken RelB transcription factor. The predicted chicken RelB protein has a high degree of sequence similarity to other vertebrate RelB proteins within the RH domain, but is much less conserved outside this domain. Chicken RelB does not bind DNA as a homodimer, but forms DNA-binding heterodimers with NF-kappaB p50 or p52. Overexpressed chicken RelB localizes to the nucleus in chicken embryo fibroblasts, and the nonconserved C-terminal sequences of chicken RelB contain a transactivation domain that functions in chicken and mouse fibroblasts. Thus, chicken RelB has functional properties similar to other vertebrate RelB proteins. However, Western blotting of diverse chicken tissues indicates that chicken RelB is more widely expressed than mammalian RelB.

Interferon regulatory factor 4 contributes to transformation of v-Rel-expressing fibroblasts.

The avian homologue of the interferon regulatory factor 4 (IRF-4) and a novel splice variant lacking exon 6, IRF-4DeltaE6, were isolated and characterized. Chicken IRF-4 is expressed in lymphoid organs, less in small intestine, and lungs. IRF-4DeltaE6 mRNA, though less abundant than full-length IRF-4, was detected in lymphoid tissues, with the highest levels observed in thymic cells. IRF-4 is highly expressed in v-Rel-transformed lymphocytes, and the expression of IRF-4 is increased in v-Rel- and c-Rel-transformed fibroblasts relative to control cells. The expression of IRF-4 from retrovirus vectors morphologically transformed primary fibroblasts, increased their saturation density, proliferation, and life span, and promoted their growth in soft agar. IRF-4 and v-Rel cooperated synergistically to transform fibroblasts. The expression of IRF-4 antisense RNA eliminated formation of soft agar colonies by v-Rel and reduced the proliferation of v-Rel-transformed cells. v-Rel-transformed fibroblasts produced interferon 1 (IFN1), which inhibits fibroblast proliferation. Infection of fibroblasts with retroviruses expressing v-Rel resulted in an increase in the mRNA levels of IFN1, the IFN receptor, STAT1, JAK1, and 2',5'-oligo(A) synthetase. The exogenous expression of IRF-4 in v-Rel-transformed fibroblasts decreased the production of IFN1 and suppressed the expression of several genes in the IFN transduction pathway. These results suggest that induction of IRF-4 expression by v-Rel likely facilitates transformation of fibroblasts by decreasing the induction of this antiproliferative pathway.

Role and regulation of Rel/NF-kappaB activity in anti-immunoglobulin-induced apoptosis in WEHI-231 B lymphoma cells.

In WEHI-231 cells, anti-immunoglobulin (anti-Ig) treatment leads to both a decrease in the DNA-binding activity of p50/c-Rel/p53 protein complexes and a transient enhancement in the DNA-binding activity of p50 homodimeric complexes. These cells subsequently undergo apoptosis. Because IkappaB-alpha plays a pivotal role in the regulation of Rel/NF-kappaB activity, we have characterized both the nature and kinetics of the expression of IkappaB-alpha following anti-Ig-induced apoptosis in WEHI-231 cells. Anti-Ig treatment of WEHI-231 cells decreased the steady-state level of IkappaB-alpha mRNA, but enhanced the stability of IkappaB-alpha, leading to an accumulation of IkappaB-alpha in both the cytosol and nucleus. Concomitant with the increase in IkappaB-alpha expression there was a gradual decline in the nuclear expression of c-Rel. Because c-Rel plays an important role in the survival of WEHI-231 cells, these results suggest that post-transcriptional regulation of IkappaB-alpha expression might play a role in the anti-Ig-induced apoptosis in WEHI-231 cells.

Degradation of proto-oncoprotein c-Rel by the ubiquitin-proteasome pathway.

The c-rel proto-oncogene product, c-Rel, belongs to the Rel/NF-kappaB transcription factor family, which regulates a large variety of cellular functions. The activation of NF-kappaB involves the degradation of the inhibitor, IkappaB, through the ubiquitin-proteasome (Ub-Pr)-mediated pathway. Here we report that the turnover of c-Rel is also regulated by the Ub-Pr pathway, thus adding another level of complexity to the regulation of NF-kappaB. High molecular weight ubiquitinated c-Rel conjugates are detected in cells and accumulate in cells treated with proteasome inhibitors. In a cell-free in vitro degradation assay, c-Rel is degraded specifically through the Ub-Pr pathway. N-terminally truncated c-Rel is readily degraded, implying the dispensability of N-terminal sequence; in contrast, a series of deletion mutants missing C-terminal sequences display a reduced susceptibility to the degradation. Interestingly, the sequence between residues 118 and 171 of c-Rel, i.e. the region immediately following the c-Rel/v-Rel homology domain, appears to play an important role in mediating ubiquitin conjugation and the subsequent degradation. Together with our previous study showing an elevated tumorigenic potential for C-terminally truncated mutants, our data suggest that the C-terminal domain of c-Rel plays an important role in mediating c-Rel degradation and growth control.

AP-1 factors play an important role in transformation induced by the v-rel oncogene.

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.

Identification of v-Rel oncogene-induced inhibitor of apoptosis by differential display.

The v-Rel oncoprotein is a member of the Rel/NF-kappaB family of transcription factors. v-Rel induces oncogenic transformation and inhibits apoptosis. To identify aberrantly expressed cellular genes in v-Rel transformed cells, gene expression patterns in normal and v-Rel transformed cells were compared by mRNA differential display. Northern blotting analysis with radiolabeled cDNAs from differential display confirmed the reproducible differential expression of 10 transcripts in v-Rel transformed cells. One of the identified genes, termed ch-IAP1, encodes a chicken homolog of the inhibitor-of-apoptosis protein (IAP) family. ch-IAP1 contains N-terminal baculovirus IAP repeats (BIR), the hallmark of IAPs, and has a C-terminal RING finger motif commonly present in the other IAPs. Like other IAPs, ch-IAP1 is expressed predominantly in the cytoplasm of cells. ch-IAP1 is highly expressed in v-Rel transformed fibroblasts, B- and T-cell lines, and spleen cell lines. In contrast, ch-IAP1 expression levels were low in chicken cell lines transformed by several other unrelated tumor viruses. Additionally, ch-IAP1 expression is downregulated in temperature-sensitive (ts) v-Rel transformed spleen cells at the nonpermissive temperature. Overexpression of the full-length ch-IAP1 suppresses mammalian cell apoptosis induced by the interleukin-1-converting enzyme (ICE), a member of the mammalian caspase family of cysteine proteases. Furthermore, expression of exogenous ch-IAP1 inhibits apoptosis of ts v-Rel transformed spleen cells at the nonpermissive temperature.

ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a mediator of the antiapoptotic activity of the v-Rel oncoprotein.

The oncoprotein v-Rel, a member of the Rel/NF-kappaB family of transcription factors, induces neoplasias and inhibits apoptosis. To identify differentially regulated cellular genes and to evaluate their relevance to transformation and apoptosis in v-Rel-transformed cells, mRNA differential display has been used. One of the recovered cDNAs corresponds to a gene that was highly expressed in v-Rel-transformed fibroblasts. Analysis of the isolated full-length cDNA of a chicken inhibitor-of-apoptosis protein (ch-IAP1) revealed that it encodes a 68-kDa protein that is highly homologous to members of the IAP family, such as human c-LAP1. Like other IAPs, ch-IAP1 contains the N-terminal baculovirus IAP repeats and C-terminal RING finger motifs. Northern blot analysis identified a 3.3-kb ch-IAP1 transcript expressed at relatively high levels in the spleen, thymus, bursa, intestine, and lungs. Expression of v-Rel in fibroblasts, a B-cell line, and spleen cells up-regulated the expression of ch-IAP1. In contrast, ch-IAP1 expression levels were low in chicken cell lines transformed by several other unrelated tumor viruses. ch-IAP1 was expressed predominantly in the cytoplasm of the v-Rel-transformed cells. ch-IAP1 suppressed mammalian cell apoptosis induced by the overexpression of the interleukin-1-converting enzyme. Expression of exogenous ch-IAP1 in temperature-sensitive v-Rel transformed spleen cells inhibited apoptosis of these cells at the nonpermissive temperature. Collectively, these results suggest that ch-IAP1 is induced during the v-Rel-mediated transformation process and functions as a suppressor of apoptosis in v-Rel-transformed cells.

c-Jun involvement in vitamin E succinate induced apoptosis of reticuloendotheliosis virus transformed avian lymphoid cells.

Previous studies have shown that treatment of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells with 10 microg/ml (18.8 microM) of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for 3 days induced approximately 50% of the cells to undergo apoptosis. Elevated and prolonged expression of c-jun mRNA and protein was temporally correlated with VES-induced cell death. Data presented in this paper show that the elevated and prolonged expression of c-jun message and protein are not accounted for by enhanced stability, and show the involvement of c-Jun in VES-induced apoptosis in this lymphoblastoid cell type. C4-1 cells infected with a virus carrying a dominant, negatively acting mutant form of c-Jun, supjun-1, exhibited: (i) 71% reduction in VES-induced apoptosis, (ii) a 2.0-2.5-fold decrease in wildtype, endogenous c-Jun expression, and (iii) a 2.4-2.6-fold reduction in AP-1 binding activity. Additionally, cells co-treated with VES plus RRR-alpha-tocopherol, exhibited a 70% reduction in apoptosis, a marked reduction in c-Jun expression and a 1.6-fold reduction in AP-1 binding activity. These studies suggest that c-Jun plays a crucial role in VES-induced apoptosis in C4-1 cells, and add to our understanding of mechanisms of action involved in VES-mediated tumor cell growth inhibition.

Differences in kappaB DNA-binding properties of v-Rel and c-Rel are the result of oncogenic mutations in three distinct functional regions of the Rel protein.

The oncogene v-rel of Reticuloendotheliosis virus, strain T, is derived from an avian c-rel proto-oncogene. c-rel encodes a member of the Rel/NF-kappaB family of transcription factors. The highly oncogenic v-Rel differs from c-Rel which has low transforming potential by the acquisition of numerous mutations. In this manuscript, we demonstrate that the oncogenic mutations in v-Rel directly alter the ability of this protein to bind to DNA. Electrophoretic mobility shift analysis with Rel proteins synthesized in vitro as well as isolated from nuclei of Rel expressing cells showed that three mutation clusters, present in the N-terminus, the center and the C-terminus of v-Rel, altered three different aspects of DNA binding. In contrast, the oncogenic C-terminal deletion of 118 amino acids present in v-Rel had almost no influence on its DNA binding. The N-terminal mutation cluster altered the kappaB DNA-binding specificity of the v-Rel oncoprotein relative to c-Rel. The mutation Met-20-->Thr was found to be principally responsible for this alteration. The second mutation cluster was responsible for increased binding of v-Rel to all the kappaB sites examined presumably because it stabilized v-Rel homodimers. This alteration in DNA binding was mapped to the group of two mutations within the cluster. In contrast, the third mutation cluster in the C-terminus of v-Rel destabilized the binding of v-Rel to all of the kappaB sites examined. This is the first indication that regions outside the Rel Homology Region can participate in the control of binding of the c-Rel protein to DNA. The three mutation clusters examined contributed to the tumorigenic potential of v-Rel with the relative strength decreasing with their position from the N-terminus to the C-terminus. These results suggest that the oncogenic mutations in v-Rel cooperate and enable v-Rel to form nuclear complexes with aberrant DNA-binding properties that may directly alter gene expression and DNA replication resulting in the transformation of the cell.

Synergistic stimulation of avian I kappa B alpha transcription by rel and fos/jun factors.

Rel/NF-kappa B transcription factors and I Kappa B alpha function in an autoregulatory network. Avian I kappa B alpha transcription is increased in response to both c-Rel and v-Rel. This study shows that I kappa B alpha transcription is synergistically stimulated by Rel and AP-1 factors (c-Fos and c-Jun). However, the response to v-Rel and the AP-1 factors was not as vigorous as that of c-Rel and AP-1. A 386 bp region of the I kappa B alpha promoter (containing two NF-kappa B and one AP-1 binding sites) was shown to be both necessary and sufficient for response to both Rel factors alone or Rel factors in conjunction with the AP-1 proteins. In addition, an imperfect NF-kappa B binding site was found to overlap the AP-1 binding site. Mutation of either of the NF-kappa B binding sites or the AP-1 binding site dramatically decreased the response of the I kappa B alpha promoter to Rel proteins alone or Rel and AP-1 factors. Overexpression of c-Rel or v-Rel resulted in the formation of DNA binding complexes associated with the imperfect NF-kappa B binding site which overlaps the AP-1 site. v-Rel associated with the imperfect NF-kappa B site stronger than c-Rel, and overexpression of v-Rel also resulted in the formation of a v-Rel containing complex bound to a consensus AP-1 site. These studies address the difference in c-Rel and v-Rel's ability to synergistically stimulate I kappa B alpha expression in conjunction with the AP-1 factors.

Inhibition of Na+K+ATPase activity in membranes of Sindbis virus-infected chick cells.

Alterations in intracellular concentrations of Na+ and K+ in Sindbis virus-infected cells result largely from inhibition of ouabain-sensitive Na+K+ATPase (Na+ pump) activity. Here we report that membrane preparations derived from Sindbis virus-infected chick cells exhibit reduced Na+K+ATPase activity, indicating that limitation of cellular factors is not responsible for inhibition of ion transport. In vitro phosphorylation of the Na+K+ATPase by [32P]orthophosphate or [gamma 32P]ATP is unaltered in membranes of Sindbis virus-infected cells, indicating that a loss of specific enzymatic functions unrelated to formation of Na+ pump phosphoenzyme intermediates occurs during the course of viral infection. However, phosphoenzyme intermediates of the Na+K+ATPase prepared from membranes of Sindbis virus-infected cells are inherently less stable than those prepared with membranes of uninfected cells. The instability of these intermediates in vitro is correlated with an altered capacity of the Na+ pump to transport monovalent cations into virus-infected cells. 22Na+ transport studies reveal enhanced ouabain-sensitive Na+ uptake by Sindbis virus-infected cells, suggesting that the Na+ pump may catalyze enhanced Na+-Na+ exchange in the infected cells. These results indicate that the capacity of the Na+K+ATPase to discriminate between binding of extracellular Na+ and K+ is specifically altered during the course of infection by Sindbis virus.

Structure and regulation of the gene encoding avian inhibitor of nuclear factor kappa B-alpha.

The Rel/NF-kappa B family of transcription factors exist in the cytoplasm as inactive complexes in association with an inhibitory protein called I kappa B-alpha. We have isolated a clone containing the avian I kappa B-alpha gene from a chicken genomic library. Avian I kappa B-alpha is devoid of any recognizable promoter elements, i.e., TATA and CAAT boxes; however, the 5'-UTR of the gene contains the initiator elements frequently found in TATA-less genes. Avian I kappa B-alpha contains seven putative Rel/NF-kappa B binding sites. A CAT reporter construct containing the 5' upstream region of I kappa B-alpha was expressed when transfected into cells which produce I kappa B-alpha. This construct, however, was not expressed in cells in which I kappa B-alpha activity was not induced, indicating that the regulatory elements which promote I kappa B-alpha expression are contained within 1000 nt of the transcription start site. Southern analysis suggests that I kappa B-alpha is present as a single-copy gene per haploid genome and is expressed in avian hematopoietic tissues, as well as lymphoid cells transformed by avian reticuloendotheliosis virus (REV-T).

Avian I kappa B alpha is transcriptionally induced by c-Rel and v-Rel with different kinetics.

The Rel/NF-kappa B family of transcription factors participates in the regulation of genes involved in defense responses, inflammation, healing and regeneration processes, and embryogenesis. The control of the transcriptional activation potential of the Rel/NF-kappa B proteins is mediated, in part, by their association with inhibitory proteins of the I kappa B family. This association results in the cytoplasmic retention of these factors until the cell receives a proper stimulatory signal. The I kappa B alpha gene is a target for regulation by the Rel/NF-kappa B proteins and is in fact upregulated in response to Rel/NF-kappa B activation. A naturally occurring oncogenic variant of the Rel/NF-kappa B family, v-rel, transforms avian lymphocytes, bone marrow cells, monocytes, and fibroblasts. Avian I kappa B alpha expression is upregulated in cells transformed by v-Rel. Avian I kappa B alpha is also upregulated in fibroblasts overexpressing c-Rel and oncogenic variants of c-Rel. c-Rel, a carboxy-terminally truncated variant of c-Rel, and v-Rel are all able to directly transactivate the expression of the avian I kappa B alpha gene. However, c-Rel was the most potent activator of this gene, and the induction of I kappa B alpha expression showed faster kinetics in cells overexpressing c-Rel than in those overexpressing v-Rel. The regulation of I kappa B alpha induction by the Rel proteins was shown to be dependent on a 362-bp region of the I kappa B alpha promoter that contains two potential NF-kappa B binding sites and one AP-1-like binding site. Results of electrophoretic mobility shift assays using these NF-kappa B binding sites indicate that major changes in the profile of DNA binding complexes in fibroblasts overexpressing v-Rel correlated temporally with the kinetic changes in v-Rel's ability to activate the expression of the I kappa B alpha gene.

Mutations in the DNA-binding and dimerization domains of v-Rel are responsible for altered kappa B DNA-binding complexes in transformed cells.

The c-rel proto-oncogene encodes a member of the Rel/NF-kappa B family of transcription factors. The oncogenic viral form, v-rel, transduced by avian reticuloendotheliosis virus T, induces lymphoid tumors. v-Rel transformation may be mediated directly by binding of v-Rel to cognate DNA sites, resulting in altered gene expression, and/or indirectly by releasing Rel/NF-kappa B transcription factors from cytoplasmic retention molecules, resulting in their translocation to the nucleus and the inappropriate expression of genes under kappa B control. v-Rel-transformed cell lines of different phenotypes contained v-Rel as well as endogenous kappa B DNA-binding proteins in nuclear extracts. Kinetic analysis with avian leukosis virus-transformed B-cell lines expressing v-Rel or c-Rel indicated that the presence of endogenous kappa B DNA-binding proteins in the nucleus is temporally correlated with the relocalization of v-Rel to the cytoplasm. Supershift analysis of these DNA-binding complexes revealed that v-Rel was present in all of the nuclear DNA-binding complexes heterodimerized with c-Rel, NF-kappa B1, and other proteins. In contrast, c-Rel-transformed cells exhibited a less-complex pattern of nuclear kappa B DNA-binding complexes, and the nuclear appearance of these endogenous complexes was not observed. Studies with c-/v-Rel hybrids suggest that the induction of the endogenous kappa B DNA-binding complexes is the result of the mutations in the C-terminal region of the Rel homology (RH) domain of v-Rel. Moreover, v-Rel differed from c-Rel in its DNA-binding specificity. The altered DNA-binding specificity of v-Rel was associated with mutations located in the N-terminal part of the RH domain of v-Rel. These results suggest that two different regions of v-Rel (both located in the RH domain) influence the formation of kappa B DNA-binding complexes differently.

The relocalization of v-Rel from the nucleus to the cytoplasm coincides with induction of expression of Ikba and nfkb1 and stabilization of I kappa B-alpha.

The v-Rel oncogene induces the expression of major histocompatibility complex class I and II proteins and the interleukin-2 receptor more efficiently than does c-Rel (R. Hrdlicková, J. Nehyba, and E. H. Humphries, J. Virol. 68:308-319, 1994). The kinetics with which these immunoregulatory receptors are induced in B- and T-lymphoid cell lines and chicken embryo fibroblast cultures expressing c-Rel or v-Rel have been examined. v-Rel induced the expression of major histocompatibility complex classes I and II and interleukin-2 receptor more efficiently than did c-Rel at later times after infection. In all three cell types, this increased efficiency was accompanied by a shift in the majority of v-Rel from the nucleus of the cytoplasm. The concomitant relocalization of v-Rel was also demonstrated during the in vitro transformation of spleen cells. The translocation coincided with increased steady-state levels of I kappa B-alpha. Coninfection by retroviral vectors expressing v-Rel, I kappa B-alpha, or NF-kappa B1 demonstrated that either I kappa B-alpha can contribute to the shift of v-Rel to the cytoplasmic compartment. The induction of nfkb1 and Ikba mRNA and the stabilization of I kappa B-alpha by v-Rel were shown to be responsible for these effects. In comparison with c-Rel, the expression of v-Rel was associated with lower levels of transcription of these genes. However, the ability of v-Rel to stabilize I kappa B-alpha remained unchanged. The ability of v-Rel to stabilize I kappa B-alpha but poorly induce Ikba mRNA expression relative to c-Rel may play a role in regulating gene expression, thereby leading to transformation.

Transformation of avian fibroblasts overexpressing the c-rel proto-oncogene and a variant of c-rel lacking 40 C-terminal amino acids.

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-Rel delta) 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 I kappa B 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 I kappa B protein pp40 and the avian homolog of the NF-kappa B protein, p105. However, expression of v-Rel resulted in the induction of pp40 mRNA only. While c-Rel was a weak activator of kappa B-mediated transcription of a reporter construct in transformed CEFs, v-Rel and c-Rel delta were transcriptional repressors. However, in spite of these differences, all of these proteins resulted in the transformation of CEFs.