Moloney murine leukemia virus-induced myeloid tumors in adult BALB/c mice: requirement of c-myb activation but lack of v-abl involvement.

BALB/c mice treated with pristane and Abelson virus have been used as an animal model system for the rapid induction of plasmacytomas. Myelomonocytic tumors with helper Moloney murine leukemia virus clonally inserted into the c-myb locus were observed in about 10% of pristane-primed BALB/c mice infected with Abelson virus. However, v-abl was absent in almost all of those tumors. Since Moloney virus is thought to induce mostly T-cell lymphomas, we have carried out studies to investigate this alteration of disease specificity and to determine whether v-abl played an obligatory role in the development of these tumors. We found that, whereas lymphomas developed late (greater than 3 months) in both pristane-primed and unprinted control mice, the myelomonocytic tumors arose at a high frequency, within 3 months, but only in pristane-treated mice. Clonal Moloney virus insertion was again found in each of the seven myelomonocytic tumors examined. Northern blot analyses and S1 mapping studies revealed the presence of virally promoted chimeric mRNAs that lack the three 5'-most myb coding exons. Hence it appears that the requirement for the v-abl gene product in tumor induction is not obligatory. Our results also indicate that tumor-specific alteration at the 5' end of the myb gene plays an important role in the development of these tumors.     

New sites of proviral integration associated with murine promonocytic leukemias and evidence for alternate modes of c-myb activation.

Murine promonocytic leukemias involving insertional mutagenesis of the c-myb locus can be induced by replication-competent retroviruses. In previously studied promonocytic leukemic cells induced by Moloney murine leukemia virus (called MML), the provirus has been invariably integrated upstream of exons 3 or 4 and the leukemic cells expressed aberrant RNAs with fused virus-myb sequences. Furthermore, Myb expressed by these cells has been shown to be truncated by 47 or 71 amino acids. The present report examines the mechanisms of myb activation in leukemias induced by two other retroviruses, amphotropic virus 4070A and Friend strain FB29 (the leukemias are called AMPH-ML and FB-ML, respectively). This study revealed two additional c-myb proviral insertion sites in these promonocytic leukemias. One FB-ML had a proviral integration in exon 9, and expressed a C-terminally truncated Myb protein of 47 kDa similar to that previously demonstrated to be expressed in the myelomonocytic cell lines NFS60 and VFL-2. However, a sequence of reverse-transcribed and amplified RNA from this leukemia demonstrated that the truncation involved a loss of 248 amino acids compared with a loss of 240 amino acids in the myelomonocytic cell lines. Another leukemia had a provirus integrated in the 5' end of c-myb upstream of exon 2 (in the first intron) and produced a Myb protein that was indistinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from normal Myb. This latter leukemia (FB-ML R1-4-10) expressed Myb with the smallest N-terminal truncation observed so far in promonocytic leukemias; translation begins at an ATG within c-myb exon 2, leading to loss of only 20 amino acids from the N terminus. Unlike the proteins produced in Moloney murine leukemia virus-induced promonocytic leukemias (MML) that have larger truncations, this protein has an intact DNA binding region and does not contain N-terminal amino acids encoded by gag. However, this protein is similar to all N-terminally truncated Mybs so far studied, in that the truncation resulted in deletion of a casein kinase II phosphorylation site which has been proposed to be involved in regulation of DNA binding.     

An immunoreceptor tyrosine activation motif in the mouse mammary tumor virus envelope protein plays a role in virus-induced mammary tumors

Mouse mammary tumor virus (MMTV) induces breast cancer with almost 100% efficiency in susceptible strains through insertional activation of protooncogenes, such as members of the wnt and fibroblast growth factor (fgf) families. We previously showed that expression of the MMTV envelope protein (Env) in normal immortalized mammary epithelial cells grown in three-dimensional cultures caused their morphological transformation, and that this phenotype depended on an immunoreceptor tyrosine-based activation motif (ITAM) present in Env and signaling through the Syk tyrosine kinase (E. Katz, M. H. Lareef, J. C. Rassa, S. M. Grande, L. B. King, J. Russo, S. R. Ross, and J. G. Monroe, J. Exp. Med. 201:431-439, 2005). Here, we examined the role of the Env protein in virus-induced mammary tumorigenesis in vivo. Similar to the effect seen in vitro, Env expression in the mammary glands of transgenic mice bearing either full-length wild-type provirus or only Env transgenes showed increased lobuloalveolar budding. Introduction of the ITAM mutation into the env of an infectious, replication-competent MMTV or into MMTV/murine leukemia virus pseudotypes had no effect on incorporation of Env into virus particles or on in vitro infectivity. Moreover, replication-competent MMTV bearing the ITAM mutation in Env infected lymphoid and mammary tissue at the same level as wild-type MMTV and was transmitted through milk. However, mammary tumor induction was greatly attenuated, and the pattern of oncogene activation was altered. Taken together, these studies indicate that the MMTV Env protein participates in mammary epithelial cell transformation in vivo and that this requires a functional ITAM in the envelope protein.     

Two functionally distinct anti-tumor effector cells isolated from primary murine sarcoma virus-induced tumors.

The ability of cells from primary MSV-induced tumors to function as effector cells in vitro was evaluated. Host cells were isolated by enzymatic disaggregation of the tumor and fractionated by sedimentation velocity at unit gravity on a Ficoll gradient. Characterization of these cells indicated that 30 to 40 % were T lymphocytes, about 50% were macrophages and less than 5% were B lymphocytes. Two different functional activities were mediated by these cells: cytolysis, as measured by the CRA, and inhibition of proliferation, as measured by the GIA. The effector cells in the CRA were T cells with sedimentation velocities of 3.5 to 4.0 mm/hr, whereas those cells which mediated the GIA were presumably macrophages and displayed a heterogeneity in size two peak sedimentation velocities, one at 4.0 mm/hr and another at 6.0 mm/hr. Activity by the effector cells in the CRA was antigen specific in contrast to the activity in the GIA which was directed against cells which did not carry detectable cross-reacting antigens.     

c-erbB2 and c-myb induce mouse oocyte maturation involving activation of maturation promoting factor

Proto-oncogenes are involved in cell growth, proliferation, and differentiation. In the present study, we investigated the roles and mediating pathways of proto-oncogenes c-erbB(2) and c-myb in mouse oocyte maturation by RT-PCR, real-time quantitative PCR, western blot, and recombinant proto-oncogene protein microinjection. Results showed that both c-erbB(2) and c-myb antisense oligodeoxynucleotides (c-erbB(2) ASODN and c-myb ASODN) inhibited germinal vesicle breakdown and the first polar body extrusion in a dose-dependent manner. However, microinjection of recombinant c-erbB(2) or c-myb protein into germinal vesicle stage oocytes stimulated oocyte meiotic maturation. In addition, the expression of c-erbB(2) and c-myb mRNA was detected in oocytes; and c-erbB(2) ASODN and c-myb ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expression, respectively. Maturation promoting factor (MPF) inhibitor roscovitine did not affect the expression of c-erbB(2) mRNA and c-myb mRNA, but blocked the effects of recombinant c-erbB(2) and c-myb protein-induced oocyte maturation. Further, cyclin B1 protein expression in oocytes was remarkably inhibited by c-erbB(2) ASODN, c-myb ASODN, and roscovitine. Nonsense tat ODN had no effect on the expression of c-erbB(2), c-myb, and cyclin B1. These results suggest that c-erbB(2) and c-myb may induce oocyte maturation through mediating a pathway involving the activation of MPF.     

Specific activation of the cellular Harvey-ras oncogene in dimethylbenzanthracene-induced mouse mammary tumors.

Genomic DNAs from dimethylbenzanthracene-induced BALB/c mouse mammary tumors arising from the transplantable hyperplastic outgrowth (HPO) line designated DI/UCD transformed NIH 3T3 cells upon transfection. Transforming activity was attributed to the presence of activated Harvey ras-1 oncogenes containing an A----T transversion at the middle adenosine nucleotide in codon 61. DNAs from untreated DI/UCD HPO cells and radiation-induced and spontaneous mammary tumors from the DI/UCD HPO line failed to transform NIH 3T3 cells. The results indicated that the mutation activation of Harvey ras-1 oncogenes was specific to dimethylbenzanthracene treatment in the mouse mammary tumor system.     

c-myb protein expression is a late event during T-lymphocyte activation.

The expression of p80c-myb was examined during the activation of resting human T lymphocytes. Before activation, no detectable p80c-myb was present. Synthesis of p80c-myb was observed only after initiation of the S phase of the cell cycle.     

Protein truncation is required for the activation of the c-myb proto-oncogene.

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.     

Characterization of the sequence-specific interaction of mouse c-myb protein with DNA.

We have examined parameters that affect sequence-specific interactions of the mouse c-myb protein with DNA oligomers containing the Myb-binding motif (CA/CGTTPu). Complexes formed between these oligomers and in vitro translated c-myb proteins were analysed by electrophoresis on non-denaturing polyacrylamide gels using the mobility-shift assay. By progressive truncation of c-myb coding sequences it was demonstrated that amino acids downstream of a region of three imperfect 51-52 residue repeats (designated R1, R2 and R3), which are located close to the amino terminus of the protein, had no qualitative or quantitative effect on the ability to interact specifically with this DNA motif. However, removal of only five amino acids of the R3 repeat completely abolished this activity. The contribution of individual DNA-binding domain repeats to this interaction was investigated by precisely deleting each individually: it was demonstrated that a combination of R2 and R3 was absolutely required for complex formation while the R1 repeat was completely dispensible. c-myb proteins showed quantitatively greater interaction with oligomers containing duplicated rather than single Myb-binding motif, in particular where these were arranged in tandem. Moreover, it was observed that c-myb protein interacted with these tandem motifs as a monomer. These findings imply that a single protein subunit straddles adjacent binding sites and the implications for c-myb activity are discussed.     

Structural organization and nucleotide sequence of mouse c-myb oncogene: activation in ABPL tumors is due to viral integration in an intron which results in the deletion of the 5'' coding sequences.

Bacteriophage libraries of mouse DNA were screened for sequences homologous to the v-myb oncogene and two overlapping clones containing the v-myb related region were isolated. Restriction enzyme mapping, heteroduplex analysis and nucleotide sequence analysis revealed the presence of nine exons. Six of these exons are homologous to the v-myb region while the other three exons are derived from the 5' region which is deleted in the viral oncogene. The sequences downstream to the sixth v-myb exon are not included in the 17 kbp of DNA sequences analyzed in this study. Comparison of the structure of the normal c-myb clone with its rearranged couterpart present in plasmacytoid lymphosarcomas revealed that the rearrangements occur in this locus as a result of viral integration. Present studies demonstrate that such a viral insertion interrupts the c-myb coding region at a region identical to that observed in the generation of the v-myb gene of avian myeloblastosis virus and results in the synthesis of mRNAs that lack the same 5' coding region.     

Differential activation of cytotoxic and suppressor T cells against syngeneic tumors in the mouse.

The cytotoxic T cell against a methylcholanthrene-induced sarcoma, S1509a, was induced in syngeneic mice by deliberate immunization with mitomycin C (MMC)-treated live tumor cells. The soluble tumor antigen (STA) extracted from the same tumor by 3 M KCl was, however, unable to induce the cytotoxic T cell upon immunization, although it was able to activate predominantly the suppressor T cell that then specifically suppressed the effect of the cytotoxic T cell against the homologous tumor. The suppressor T cell generated by STA had the same characteristics as those found in tumor-bearing animals: 1) The suppressor T cell has a very strict specificity against individual tumors; 2) The cell expresses cell surface determinants controlled by genes in the I-J subregion of the mouse H-2 complex. The activity of the cytotoxic T cell was completely inhibited by live tumor cells but not by STA, whereas that of the suppressor T cell was neutralized by STA. The results that cytotoxic and suppressor T cells are activated under different conditions, and that the antigenic determinants recognizable by these two cell types are not the same. The soluble extract contains only the determinants recognizable by the suppressor T cell, and the cytotoxic T cell can be activated only by the determinants associated with self antigen present on the surface of live tumor cells.     

Host genetic background effect on the frequency of mouse mammary tumor virus-induced rearrangements of the int-1 and int-2 loci in mouse mammary tumors.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.     

Retroviral insertional activation of the c-myb proto-oncogene in a Marek's disease T-lymphoma cell line.

Marek's disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5' long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line.