Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements.

We describe the development of metallothionein-based vectors with low basal levels of expression that are hyperinducible upon treatment with heavy metals. Vectors were constructed by substituting a region in the hMTIIA promoter (bp -70 to -129) containing an element (BLE) involved in basal level expression with multiple metal responsive elements (MREs). In expression studies utilizing cat as a reporter gene, heavy metal inducibility was examined in both transiently transfected and permanently transformed Chinese hamster ovary (CHO) cells. Our results demonstrate that, within the same promoter structure, inducibility can be increased by altering the ratio of MREs to BLEs. Optimal induction of expression in permanently transformed CHO cells was achieved by exposure to heavy metals for 48 h prior to cell harvest, with an additional boost 12 h before harvest. These vectors have the potential to be used for production of proteins in cultured mammalian cells and in gene expression in transgenic animals.

Murine erythroid cell lines derived with c-myc retroviruses respond to leukemia-inhibitory factor, erythropoietin, and interleukin 3.

The transforming potential of the c-myc gene is shown here, for the first time, to include murine erythroid cells. Continuously growing cell lines were reproducibly generated by infection of day 13 CBA fetal liver cells with novel recombinant c-myc retroviruses. By cytostaining, most cells resembled early erythroblasts, but certain lines also contained significant numbers of hemoglobinized cells. RNA analysis revealed substantial expression of the genes encoding beta-globin and the erythroid-specific transcription factor GF-1. Although apparently immortal, the lines were not initially transplantable. Thus, constitutive myc expression in early erythroid cells can enhance their self-renewal capacity but is insufficient to fully transform them. The cell lines proliferated without the addition of exogenous factors, but their clonogenicity in semisolid medium was enhanced in the presence of erythropoietin, interleukin 3, and/or leukemia-inhibitory factor. In combination with either interleukin 3 or erythropoietin, leukemia-inhibitory factor also facilitated differentiation of certain lines. These results suggest that leukemia-inhibitory factor may have a previously unsuspected role in the regulation of erythropoiesis and could be considered as a possible therapeutic agent for the clinical management of erythroleukemia.

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.

The transgenic window on lymphoid malignancy.

Transgenic mice bearing an oncogene targetted for expression in a specific tissue can reveal how that oncogene influences differentiation and help to delineate the pathways to malignancy. To explore lymphoid neoplasia, we have made strains of transgenic mice bearing different oncogenes driven by the immunoglobulin heavy chain enhancer (E mu), which promotes expression within lymphocytes and certain myeloid cells. The prototype E mu-myc mice succumb to pre-B and B cell lymphomas, following a preneoplastic phase in which cycling pre-B cells are overproduced. The similar fate of E mu-N-myc mice suggests that N-myc and myc have overlapping functions. Surprisingly, E mu-N-ras mice develop T lymphomas and macrophage tumours but no B lineage tumours; thus the ability of ras to initiate tumorigenesis may be lineage specific. Similarly, the high predisposition of E mu-v-abl mice to develop plasmacytomas may indicate that v-abl is oncogenic only at certain stages of B cell maturation. The bcl-2 gene promotes cell survival rather than proliferation, and E mu-bcl-2 mice produce copious resting B lymphocytes. The random onset and monoclonality of tumours in the transgenic strains argues for spontaneous genetic alterations that cooperate with the trans-oncogene. Indeed, most plasmacytomas of E mu-v-abl mice bear spontaneous myc rearrangements. Moreover, a minority of E mu-myc B lymphomas exhibit ras mutation, and the tumorigenesis can be reconstructed by crossing E mu-myc and E mu-ras mice, or by retroviral delivery of v-ras or v-raf, either in vitro or in vivo. To access novel cooperating oncogenes, we are using a retrovirus lacking an oncogene as an insertional mutagen. This approach should be applicable to any trans-oncogenic strain and help to delineate the genetic events that trigger malignant clones.