Oncogene cooperation in lymphocyte transformation: malignant conversion of E mu-myc transgenic pre-B cells in vitro is enhanced by v-H-ras or v-raf but not v-abl.

Although transgenic mice bearing a c-myc gene controlled by the immunoglobulin heavy-chain enhancer (E mu) eventually develop B-lymphoid tumors, B-lineage cells from preneoplastic bone marrow express the transgene but do not grow autonomously or produce tumors in mice. To determine whether other oncogenes can cooperate with myc to transform B-lineage cells, we compared the in vitro growth and tumorigenicity of normal and E mu-myc bone marrow cells infected with retroviruses bearing the v-H-ras, v-raf, or v-abl oncogene. The v-H-ras and v-raf viruses both generated a rapid polyclonal expansion of E mu-myc pre-B bone marrow cells in liquid culture and 10- to 100-fold more pre-B lymphoid colonies than normal in soft agar. The infected transgenic cells were autonomous, cloned efficiently in agar, and grew as tumors in nude mice. While many pre-B cells from normal marrow could also be induced to proliferate by the v-raf virus, these cells required a stromal feeder layer, did not clone in agar, and were not malignant. Most normal cells stimulated to grow by v-H-ras also cloned poorly in agar, and only rare cells were tumorigenic. With the v-abl virus, no more cells were transformed from E mu-myc than normal marrow and the proportion of tumorigenic pre-B clones was not elevated. These results suggest that both v-H-ras and v-raf, but apparently not v-abl, collaborate with constitutive myc expression to promote autonomous proliferation and tumorigenicity of pre-B lymphoid cells.     

Immunologic competence of B cells subjected to constitutive c-myc oncogene expression in immunoglobulin heavy chain enhancer myc transgenic mice

B cell activation is associated with a marked transient rise in expression of the c-myc proto-onco-gene. A unique opportunity to examine the effects of constitutive c-myc expression upon B cell function is provided by transgenic mice in which the c-myc oncogene is regulated by the enhancer (E mu) from the immunoglobulin locus (E mu-myc mice). We have examined the immunologic competence of B cells from E mu-myc mice both in vitro and in vivo. Upon stimulation, many E mu-myc B cells can proliferate to form clones most of which contain antibody-forming cells. However, the frequency of responsive B cells from E mu-myc donors is only about 30% that of B cells from normal littermates. Thus, enforced myc expression is not sufficient to block the differentiation of all B cells, but a much larger fraction of the immunoglobulin-bearing cells from E mu-myc mice are incompetent. Upon immunization, E mu-myc mice mounted specific antibody responses, although some responses were delayed. Isotype switching can occur, since we observed hemolytic plaques of both IgM and IgG type and detected specific antibody of both classes in the serum. Moreover, the serum from nonimmunized E mu-myc mice contained normal levels of both IgM and IgG. Thus constitutive expression of the c-myc gene appears to retard B cell differentiation, but does not grossly impair immunologic function in the intact animal.     

The c-myc oncogene perturbs B lymphocyte development in E-mu-myc transgenic mice

Transgenic mice bearing a c-myc oncogene subjugated to the lymphoid-specific immunoglobulin heavy chain enhancer (E mu) develop clonal B lymphoid malignancies, but most young E mu-myc mice lack malignant clones. Their prelymphomatous state has allowed us to examine how constitutive c-myc expression influences B cell development. We find that early stages are overrepresented, even before birth. Pre-B cells of polyclonal origin increase greatly, while B cells develop in reduced number. Both the pre-B and the B cells appear to be in an active state, since they are larger than normal and a greater fraction are in the cell cycle. Enforced myc expression has thus favored proliferation over maturation. Hence, a normal function of c-myc may be to regulate differentiation as well as to promote cell cycling.     

An E mu-v-abl transgene elicits plasmacytomas in concert with an activated myc gene.

To clarify how the v-abl oncogene of Abelson murine leukemia virus contributes to lymphoid tumorigenesis, we introduced the gene linked to an immunoglobulin heavy chain enhancer (E mu) into the mouse germline. Although lymphoid development was not detectably affected in young E mu-v-abl mice, three transgenic lines shared a high predisposition to develop clonal plasmacytomas that secreted IgA or IgG. The unexpected absence of pre-B lymphomas suggests that Abelson virus generates such tumors by infecting an early lymphoid progenitor cell that has not yet activated the heavy chain enhancer. Most plasmacytomas bore a rearranged c-myc gene, apparently as a result of spontaneous translocation to the Igh locus. Moreover, progeny of a cross with analogous E mu-myc mice rapidly developed oligoclonal plasmacytomas. Thus, the collusion of v-abl with c-myc is stage specific, efficiently transforming plasma cells but not pre-B cells or B cells.     

N-myc transgene promotes B lymphoid proliferation, elicits lymphomas and reveals cross-regulation with c-myc.

To assess the impact of constitutive N-myc expression on lymphocytes, we generated lines of transgenic mice bearing the murine N-myc oncogene coupled to the immunoglobulin heavy chain enhancer (E mu). As in mice carrying an analogous c-myc construct, E mu-N-myc mice exhibit a limited overgrowth of cycling pre-B cells and eventually succumb to clonal B lymphoid tumours. The endogenous N-myc and c-myc alleles are silent in both E mu-N-myc and E mu-myc lymphomas, suggesting that these genes are subject to auto- and cross-regulation. The regulatory interaction and the similar biological effects of N-myc and c-myc imply that the two genes perform interchangeable functions in the promotion of cell proliferation.     

Novel zinc finger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in E mu-myc transgenic mice

To search for genes that can collaborate with myc in lymphomagenesis, we exploited retroviral insertional mutagenesis in E mu-myc transgenic mice. Moloney murine leukemia virus accelerated development of B lymphoid tumors. Three quarters contained a provirus within the known pim-1 or pim-2 loci, new loci bmi-1 and emi-1, or combinations of these. bmi-1 insertions predominated, occurring in half the tumors, and resulted in elevated bmi-1 mRNA levels. Significantly, the bmi-1 gene, which is expressed in diverse normal cells, encodes a Cys/His metal-binding motif (C3HC4) that resembles those in several DNA-binding proteins and defines a new category of zinc finger gene. Thus, myc-induced lymphomagenesis can entail the concerted action of several genes, including the presumptive nuclear regulator bmi-1.     

In situ study of c-myc protein expression during avian development

The distribution of the c-myc protein was studied in the developing embryo from the two-somite stage to embryonic day 17 (E17). A triple labelling method was used, with a polyclonal serum recognizing the human and avian c-myc proteins as the first marker followed by Hoechst 33258 for nuclear staining and the monoclonal antibody 13F4 which reveals the avian myogenic lineage. In situ hybridization was carried out at three selected stages (E3, E6 and E8), in order to compare the distribution of myc mRNA and myc protein. The c-myc protein signal was barely detectable in blastodisc nuclei during the period of somite formation, after which it became ubiquitous in the embryonic body until E4. Myotomal cell nuclei displayed a strong signal until their organization into premuscular masses. On day 4, the level of c-myc protein decreased in all embryonic tissues. By doubling the antibody titre and amplifying the signal by means of the streptavidin-biotin method, c-myc could still be detected in nuclei of defined groups of cells. Such was the case in some mesenchyme-derived tissues at critical periods of organogenesis, for instance in prechondrogenic condensations or hemopoietic cell foci at E6, the latter becoming negative at E9. The heart ventricle displayed a patch-work of positive and negative nuclei from E6 to E10. A myc signal restricted to the quail species was found in the wall of the carotid arteries. Cell nuclei in the nervous system displayed a detectable signal which became restricted to postmitotic neurones. In the ectoderm, the c-myc protein was generally not present after E4, except in presumptive feather buds at the time of epitheliomesenchymal interactions. Endodermal cells (such as hepatocytes, oesophageal and tracheal epithelia) did not express detectable levels of c-myc at any time. Our results reveal a time- and tissue-specific expression of c-myc during avian development. It is noteworthy that the expression of the c-myc protein often appears dissociated from cell proliferation as shown by the absence of the signal in endodermal cells at E3-E13 as well as its presence in postmitotic neurones. Finally, although RNA and protein are simultaneously detected in some structures such as presumptive feather buds, their expression is dissociated in endodermal tissues, notably hepatocytes, where in situ hybridization detects a large number of RNA copies with no detectable protein signal.     

Surface IgM mediated regulation of RAG gene expression in E mu-N-myc B cell lines.

Transgenic mice carrying either the c-myc or N-myc oncogene deregulated by the immunoglobulin heavy chain enhancer element (E mu) develop both pre-B and B cell lymphomas (E mu-c-myc and E mu-N-myc lymphomas). We report here that B cell lines derived from these tumors, as well as a line derived from v-myc retroviral transformation, simultaneously express surface immunoglobulin (a hallmark of mature B cells) as well as a common subset of genes normally restricted to the pre-B stage of development-including the recombinase activating genes RAG-1 and RAG-2. Continued RAG-1 and RAG-2 expression in these lines is associated with VDJ recombinase activity detected with a VDJ recombination substrate. Cross-linking of the surface immunoglobulin on these lines with an anti-mu antibody leads to rapid, specific and reversible down-regulation of RAG-1 and RAG-2 gene expression. We also find that a small but significant percentage of normal surface immunoglobulin bearing bone marrow B cells express the RAG-1 gene. These findings are discussed in the context of their possible implications for the control of specific gene expression during the pre-B to B cell transition.