Myb proteins inhibit fibroblast transformation by v-Rel
© Fu et al; licensee BioMed Central Ltd. 2006
Received: 02 November 2005
Accepted: 02 November 2006
Published: 02 November 2006
Genes that cause cancer have been divided into two general classes – oncogenes that act in a dominant fashion to transform normal cells into a malignant state, and tumor suppressor genes that act in a dominant fashion to prevent such transformation. In this report, we demonstrate that both the v-myb retroviral oncogene, which causes leukemic transformation of hematopoietic cells, and the c-myb proto-oncogene can also function as inhibitors of fibroblast transformation by the v-rel oncogene. These results imply that the myb genes can function either as oncogenes or as tumor suppressors in different cellular contexts.
The oncogenic transformation of normal cells of vertebrates is a multi-step process in which mutations accumulate in two classes of cellular genes, oncogenes and tumor suppressor genes . Oncogenes are altered forms of normal cellular proto-oncogenes that act in a dominant fashion to convert normal cells into a malignant state. In contrast, tumor suppressor mutants act in a recessive fashion within the cell and, in general, one wild type copy of a tumor suppressor gene is sufficient to inhibit transformation.
The v-myb oncogene of the avian myeloblastosis virus is unusual because unlike other known oncogenes, it causes only leukemias in animals and transforms only hematopoietic cells and not fibroblasts in culture . Members of the Myb protein family bind to specific DNA sequences, can directly regulate gene expression, and have been highly conserved during eukaryotic evolution . The myb oncogene has previously been shown to cooperate with the v-ets oncogene in the transformation of hematopoietic cells . Indeed, the ets gene family was initially discovered because of the presence of both v-myb and v-ets within a single acutely transforming retrovirus, the E26 leukemia virus .
The v-rel oncogene of the avian reticuloendotheliosis virus strain T (REV-T) causes a malignant proliferation of immature lymphoid cells in animals and can transform both lymphoid and fibroblastic cells in culture . However, fibroblast transformation by this virus is somewhat weaker than that caused by a variety of other oncogenes . Members of the Rel protein family include Drosophila Dorsal and vertebrate NF-kB, and like Myb, these proteins bind to specific DNA sequences and can directly regulate gene expression . In order to test whether v-myb and c-myb could cooperate with v-rel in oncogenic transformation of hematopoietic cells, we constructed a series of avian retroviruses that coexpress either one or both of these oncogenes. Quite unexpectedly, we found that v-myb and c-myb suppress fibroblast transformation by v-rel.
Results and discussion
v-Myb and c-Myb inhibit fibroblast transformation by v-Rel-ER
Somewhat surprisingly, the virus that encoded both the v-Rel-ER and v-Myb proteins was incapable of causing fibroblast transformation either in the presence or absence of estrogen (Figure 2). The v-Myb protein is a doubly truncated form of the normal c-Myb protein that has also sustained a number of amino acid substitutions relative to c-Myb . In order to determine whether the ability of v-Myb to suppress fibroblast transformation by v-Rel is a result of these alterations in the v-Myb protein, we constructed similar viruses that expressed either c-Myb alone, or both v-Rel-ER and c-Myb (Figure 1). As was observed with v-Myb, c-Myb itself was incapable of transforming chicken embryonic fibroblasts (not shown). Furthermore, c-Myb was able to completely suppress transformation by v-Rel-ER in a fashion similar to v-Myb (Figure 2). These results demonstrate that the v-myb gene, which is capable of oncogenically transforming macrophage precursors and causing monoblastic leukemias in vivo, can act in an opposing fashion by suppressing fibroblast transformation by the v-rel oncogene. Furthermore, the c-myb gene which can also cause the outgrowth of myelomonocytic cells in culture [15, 16], behaves similar to v-myb in suppressing transformation by v-rel.
Myb proteins inhibit the actin cable reorganization induced by v-Rel-ER
Protein expression by the dicistronic Rel/Myb viruses
Rel and Myb proteins are associated with one another in nuclear extracts
Myb genes as inhibitors of oncogenic transformation
Our results demonstrate that v-myb and c-myb, which can both cause the transformation of hemayopoietic cells, can also function as suppressors of fibroblast transformation by the v-rel oncogene. These results suggest that the cell type in which v-myb or c-myb is expressed appears to determine whether they function as oncogenes or tumor suppressor genes. Because neither v-Myb nor c-Myb are normally present in fibroblasts, one hypothesis is that v-Myb and c-Myb may act as tumor suppressors by dominantly inhibiting the function of B-Myb, a closely related protein which appears to be ubiquitously expressed in all vertebrate cells including in fibroblasts .
However, v-Myb and c-Myb do not appear to function as general suppressors of fibroblast transformation. Rather, the mechanism of transformation also plays a role in determining whether Myb proteins function as tumor suppressors, because similar experiments have shown that fibroblast transformation by the v-myc oncogene of the MC29 virus is not suppressed by the v-Myb or c-Myb proteins. Furthermore, the myb and rel oncogenes are not mutually antagonistic in all cells types. In particular, when hematopoietic yolk sac or bone marrow hematopoietic cells are infected with viruses containing both v-myb and v-rel-ER, transformed myeloid cells grow out that are indistinguishable from cells transformed by v-myb alone (data not shown).
A model that could explain these data is that v-Rel and the Myb proteins oppose each other in regulating a common set of genes that are essential for transformation by v-rel, whereas the genes essential for transformation by v-myc are not affected by Myb proteins. In this regard, gene expression profiling of transformed lymphocytes have suggested that at least some genes may be regulated in common by both Myb and Rel proteins, although many are not . Our data may also explain why v-myb is unique among the known retroviral oncogenes in that it does not transform fibroblasts – because in fibroblasts v-myb appears to function as an inhibitor of transformation rather than as an oncogene. In addition, our findings may offer some explanation for the paradoxical observation that elevated levels of c-myb proto-onoocgene expression are a positive prognostic indicator in human breast cancer .
The v-myb oncogene was discovered because of its ability to cause monoblastic leukemia in chickens. Altered forms of the c-myb proto-oncogene cause leukemia and lymphoma in birds and mammals. However, neither v-myb nor c-myb have been shown to oncogenically transform fibroblasts. We report here that v-myb and c-myb can inhibit fibroblast transformation by the v-rel oncogene, demonstrating that in at least some cellular contexts, v-myb and c-myb can function as tumor suppressors.
The construction of the N-Cla and N-ICB proviruses has been described previously [16, 24]. The N-IVB provirus was constructed by cloning the ClaI-resistant IRES-v-myb fragment of SP73-IVB into N-Cla. SP73-IVB itself contains the EcoRI/MscI-resistant IRES fragment of the murine encephalomyocarditis virus (EMCV) and the MscI/XbaI resistant v-myb fragment of MT7-MYB in a modified SP73 vector in which the entire polylinker was replaced by a single ClaI site [9, 25]. The N-Rel-ER provirus was constructed by cloning the small ClaI-resistant fragment of RCAS-Rel-ER  into the ClaI site of a modified N-dGE vector  in which the v-myb coding sequence but not the splice acceptor site had been removed by digestion with KpnI and ClaI, fill-in with the Klenow fragment of DNA polymerase I and insertion of a ClaI linker. The N-Rel plasmid was constructed in a similar fashion using the small ClaI-resistant fragment of RCAS-REL . The N-Rel-ER-IVB and N-Rel-ER-ICB proviruses were created by ligation of the small ClaI-resistant fragments from N-IVB (v-myb) or N-ICB (c-myb) into the unique ClaI site of N-Rel-ER which lies downstream of the Rel-ER open reading frame.
Cell culture and DNA transfections
Primary chicken embryo fibroblasts (CEF) were prepared by trypsinizing the bodies of 7 to 9 day old chicken embryos. These cells were grown in a 37°C, humidified 5% CO2 incubator, in Dulbecco's modified essential medium (DMEM) supplemented with glucose (4.5 g/l), 1X MEM nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, streptomycin (100 ug/ml), penicillin (100 U/ml), 2% heat-inactivated chicken serum (56°C, 1 hr), and 8% fetal calf serum. QT6 cells were grown in similar conditions except that 5% fetal calf serum and no chicken serum were added. Where indicated, estradiol was present at a concentration of 1 uM. DNA transfections, luciferase assays, and β-galactosidase assays were performed as described previously .
Actin cable staining
CEFs grown in chamber slides were washed with PBS twice and fixed with lysine-paraformaldehyde-PBS (0.075 M lysine, 0.0375 M sodium phosphate, 2% paraformaldehyde, pH 7.4) for 20 minutes at room temperature. Fixed cells were then washed three times with PBS and stained with rhodamine-phalloidin (Molecular Probes, Inc) for 25 minutes at room temperature. After staining, the cells were washed three times with PBS and overlaid with p-phenylendiamine (PPD)-mounting medium (1%(w/v) PPD, 90 % glycerol, pH 8.5) and visualized by fluorescence microscopy.
Cells were washed once with PBS, scraped off the plates in PBS, centifuged, then lysed in 1X sodium dodecyl sulfate (SDS)-loading buffer and boiled for 5 minutes. Normalized volumes of lysates were subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membranes (BA-S 83, Schleicher & Schuell). Myb expression was detected using a mixture of the Myb 2.2 and 2.7 monoclonal murine antibodies . Rel expression was detected using a polyclonal rabbit antiserum SB66 (1:1000 dilution) kindly provided by Amy Walker and Paula Enrietto. Blots were developed using either goat anti-mouse IgG or goat anti-rabbit IgG conjugated to alkaline phosphotase (Promega), 5-bromo-4-chloro-3 indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) according to the manufacturer's instructions.
Quail fibroblasts co-transfected with expression vectors for v-Rel-ER or v-Rel and the indicated Myb proteins were lysed in RIPA buffer without SDS, immunoprecipitated with either mouse the monclonal Myb 2.2 antibody which recognizes a region of v-Myb and c-Myb near the transcriptional activation domain, or the rabbit polyclonal anti-Rel antiserum SB66 using RIPA buffer without SDS and protein G sepharose . Precipitates were then analyzed by SDS-PAGE and immunoblotting using the monoclonal 5E anti-Myb antibody which recognizes the DNA binding domain . A sample of the total extract prior to immunoprecipitation was co-electrophoresed as a control.
We thank the members of our laboratory for helpful discussions. Paula Enrietto, Tom Gilmore, Tom Parson, Jonathan Sleeman, and Amy Walker kindly provided various reagents used in this study. This work was supported USPHS grant R01 CA43592 (J.S.L.).
- Hahn WC, Weinberg RA: Modelling the molecular circuitry of cancer. Nat Rev Cancer. 2002, 2: 331-341. 10.1038/nrc795View ArticlePubMedGoogle Scholar
- Lipsick JS, Wang DM: Transformation by v-Myb. Oncogene. 1999, 18: 3047-3055. 10.1038/sj.onc.1202745View ArticlePubMedGoogle Scholar
- Ganter B, Lipsick JS: Myb and oncogenesis. Adv Cancer Res. 1999, 76: 21-60.View ArticlePubMedGoogle Scholar
- McNagny KM, Graf T: Acute avian leukemia viruses as tools to study hematopoietic cell differentiation. CurrTopMicrobiolImmunol. 1996, 212: 143-162.Google Scholar
- Leprince D, Gegonne A, Coll J, de Taisne C, Schneeberger A, Lagrou C, Stehelin D: A putative second cell-derived oncogene of the avian leukaemia retrovirus E26. Nature. 1983, 306: 395-397. 10.1038/306395a0View ArticlePubMedGoogle Scholar
- Gilmore TD: The Re1/NF-kappa B/I kappa B signal transduction pathway and cancer. Cancer Treat Res. 2003, 115: 241-265.View ArticlePubMedGoogle Scholar
- Moore BE, Bose HR: Expression of the v-rel oncogene in reticuloendotheliosis virus-transformed fibroblasts. Virology. 1988, 162: 377-387. 10.1016/0042-6822(88)90478-3View ArticlePubMedGoogle Scholar
- Aggarwal BB: Nuclear factor-kappaB: the enemy within. Cancer Cell. 2004, 6: 203-208. 10.1016/j.ccr.2004.09.003View ArticlePubMedGoogle Scholar
- Jang SK, Davies MV, Kaufman RJ, Wimmer E: Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. Journal of Virology. 1989, 63: 1651-1660.PubMed CentralPubMedGoogle Scholar
- Ghattas IR, Sanes JR, Majors JE: The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Molecular and Cellular Biology. 1991, 11: 5848-5859.PubMed CentralPubMedGoogle Scholar
- Lipsick JS, Ibanez CE, Baluda MA: Expression of molecular clones of v-myb in avian and mammalian cells independently of transformation. J Virol. 1986, 59: 267-275.PubMed CentralPubMedGoogle Scholar
- Perbal B, Lipsick JS, Svoboda J, Silva RF, Baluda MA: Biologically active proviral clone of myeloblastosis-associated virus type 1: Implications for the genesis of avian myeloblastosis virus. Journal of Virology. 1985, 56: 240-244.PubMed CentralPubMedGoogle Scholar
- Boehmelt G, Walker A, Kabrun N, Mellitzer G, Beug H, Zenke M, Enrietto PJ: Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression. EMBO Journal. 1992, 11: 4641-4652.PubMed CentralPubMedGoogle Scholar
- Baluda MA, Reddy EP: Anatomy of an integrated avian myeloblastosis provirus: structure and function. Oncogene. 1994, 9: 2761-2774.PubMedGoogle Scholar
- Ferrao P, Macmillan EM, Ashman LK, Gonda TJ: Enforced expression of full length c-Myb leads to density-dependent transformation of murine haemopoietic cells. Oncogene. 1995, 11: 1631-1638.PubMedGoogle Scholar
- Fu SL, Lipsick JS: Constitutive expression of full-length c-Myb transforms avian cells characteristic of both the monocytic and granulocytic lineages. Cell Growth Differ. 1997, 8: 35-45.PubMedGoogle Scholar
- Morrison LE, Boehmelt G, Beug H, Enrietto PJ: Expression of v-rel in a replication competent virus: transformation and biochemical characterization. Oncogene. 1991, 6: 1657-1666.PubMedGoogle Scholar
- Richardson PM, Gilmore TD: vRel is an inactive member of the Rel family of transcriptional activating proteins. J Virol. 1991, 65: 3122-3130.PubMed CentralPubMedGoogle Scholar
- Sarkar S, Gilmore TD: Transformation by the vRel oncoprotein requires sequences carboxy-terminal to the Rel homology domain. Oncogene. 1993, 8: 2245-2252.PubMedGoogle Scholar
- Gill G, Ptashne M: Negative effect of the transcriptional activator GAL4. Nature. 1988, 334: 721-724. 10.1038/334721a0View ArticlePubMedGoogle Scholar
- Joaquin M, Watson RJ: Cell cycle regulation by the B-Myb transcription factor. Cell Mol Life Sci. 2003, 60: 2389-2401. 10.1007/s00018-003-3037-4View ArticlePubMedGoogle Scholar
- Neiman PE, Grbic JJ, Polony TS, Kimmel R, Bowers SJ, Delrow J, Beemon KL: Functional genomic analysis reveals distinct neoplastic phenotypes associated with c-myb mutation in the bursa of Fabricius. Oncogene. 2003, 22: 1073-1086. 10.1038/sj.onc.1206070View ArticlePubMedGoogle Scholar
- Guerin M, Sheng ZM, Andrieu N, Riou G: Strong association between c-myb and oestrogen-receptor expression in human breast cancer. Oncogene. 1990, 5: 131-135.PubMedGoogle Scholar
- Dini PW, Lipsick JS: Oncogenic truncation of the first repeat of c-Myb decreases DNA binding in vitro and in vivo. Mol Cell Biol. 1993, 13: 7334-7348.PubMed CentralPubMedGoogle Scholar
- Grasser FA, LaMontagne K, Whittaker L, Stohr S, Lipsick JS: A highly conserved cysteine in the v-Myb DNA-binding domain is essential for transformation and transcriptional trans-activation. Oncogene. 1992, 7: 1005-1009.PubMedGoogle Scholar
- Dini PW, Eltman JT, Lipsick JS: Mutations in the DNA-binding and transcriptional activation domains of v-Myb cooperate in transformation. J Virol. 1995, 69: 2515-2524.PubMed CentralPubMedGoogle Scholar
- Fu SL, Lipsick JS: FAETL motif required for leukemic transformation by v-Myb. J Virol. 1996, 70: 5600-5610.PubMed CentralPubMedGoogle Scholar
- Evan GI, Lewis GK, Bishop JM: Isolation of monoclonal antibodies specific for products of avian oncogene myb. Mol Cell Biol. 1984, 4: 2843-2850.PubMed CentralPubMedGoogle Scholar
- Harlow E, Lane DP: Antibodies: A laboratory manual. 1988, Cold Spring Harbor, Cold Spring Harbor Laboratory.Google Scholar
- Sleeman JP: Xenopus A-myb is expressed during early spermatogenesis. Oncogene. 1993, 8: 1931-1941.PubMedGoogle Scholar
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