Achim Leutz

v-erbA specifically suppresses transcription of the avian erythrocyte anion transporter (Band 3) gene

Previous work has established that the v-erbA oncogene inhibits the temperature-induced differentiation of chick erythroblasts transformed with temperature-sensitive oncogene mutants. Here we demonstrate that v-erbA in differentiating erythroblasts specifically arrests expression of the erythrocyte anion transporter (band 3) gene at the transcriptional level. The v-erbA-induced differentiation block can be overcome by inducing cells to differentiate at alkaline pH. Under these conditions, which possibly impair biological activity of v-erbA, the maturing cells now express the anion transporter gene at high levels. However, its transcription is specifically and rapidly suppressed if v-erbA activity is restored by culturing the cells at neutral pH. Similar but less pronounced inhibition of gene expression by v-erbA was observed for the delta-amino-levulinic acid synthase gene. Additional evidence obtained with an inhibitor of band 3 activity suggests that the v-erbA-induced inhibition of band 3 gene expression is at least partly responsible for the differentiation block caused by this oncogene.

Autocrine growth induced by src-related oncogenes in transformed chicken myeloid cells

Chicken myeloid cells transformed by the v-myb-or v-myc-containing leukemia viruses, E26 and OK 10, respectively, require chicken myelomonocytic growth factor (cMGF) for proliferation in vitro. Upon superinfection with retroviruses carrying oncogenes of the src gene family, these myeloid cells acquire the ability to grow in the absence of exogenous cMGF. Conditioned medium prepared from superinfected E26 cells contains a growth-stimulating activity similar in biological and immunological properties to cMGF. This activity is reduced by more than 80% following absorption of conditioned media with antiserum against cMGF. Incubation of superinfected E26 cells with an immunoglobulin fraction of antiserum against cMGF inhibits their proliferation, indicating that the cells are dependent on the secreted factor. We conclude that viral oncogenes of the src family can induce chicken myeloid cells to produce a cMGF-like factor(s) that stimulates proliferation of these cells in an autocrine fashion.

NF-M (chicken C/EBP beta) induces eosinophilic differentiation and apoptosis in a hematopoietic progenitor cell line.

CAAT/enhancer binding proteins (C/EBPs) are transcriptional activators implicated in the differentiation processes of various cell lineages. We have shown earlier that NF‐M, the chicken homolog of C/EBP beta, is specifically expressed in myelomonocytic and eosinophilic cells of the hematopoietic system. To investigate the role of NF‐M in hematopoietic cell lineage commitment, we constructed a conditional form of the protein by fusing it to the hormone binding domain of the human estrogen receptor. This construct was stably expressed in a multipotent progenitor cell line transformed by the Myb‐Ets oncoprotein. We report here that both NF‐M‐dependent promoter constructs and resident genes could be activated by addition of beta‐estradiol to the NF‐M‐estrogen receptor expressing progenitors. At the same time, we observed a down‐regulation of progenitor‐specific surface markers and the up‐regulation of differentiation markers restricted to the eosinophil and myeloid lineages. In addition to the onset of differentiation, cell death was induced with typical apoptotic features. Our results suggest that NF‐M plays an important role in commitment along the eosinophil lineage and in the induction of apoptosis.

Purification and characterization of cMGF, a novel chicken myelomonocytic growth factor.

We describe the purification of a novel hematopoietic growth factor from conditioned medium of a transformed macrophage cell line. The factor, termed chicken myelomonocytic growth factor (cMGF) stimulates the growth of chicken myeloblasts transformed by myb oncogene‐containing retroviruses and induces the formation of macrophage colonies in uninfected chick bone marrow cultures. The biological activity of the factor is destroyed by trypsin and by reducing reagents but not by SDS. Analysis of crude conditioned medium on non‐reducing SDS gels reveals two active species of cMGF with mol. wts. of 23 and 27 kd. Incubation of radioiodinated partially purified cMGF with myeloblasts demonstrates the specific binding of 23‐ and 27‐kd components under non‐reducing, and 25‐ and 29‐kd components under reducing conditions. Glycosylation inhibition experiments indicate that the larger molecules represent glycosylated forms of a single protein moiety. The 27‐kd species has been purified to homogeneity (80 000‐fold enrichment) and exerts its half maximal activity at 2 X 10(‐12) M and its maximal activity at 3 X 10(‐11) M. Antibodies prepared to purified cMGF completely neutralize the growth‐stimulating activity of the factor.

Ts mutants of E26 leukemia virus allow transformed myeloblasts, but not erythroblasts or fibroblasts to differentiate at the nonpermissive temperature

The myb, ets-containing avian acute leukemia virus E26 transforms myeloblasts, erythroblasts, and fibroblasts in culture and causes a mixed erythroid/myeloid leukemia in chicks. We report the isolation and characterization of four E26 mutants that are temperature-sensitive (ts) for myeloblast transformation. At the permissive temperature, tsE26-transformed myeloid cells resemble macrophage precursors and proliferate rapidly, provided the growth medium contains chicken myelomonocytic growth factor (cMGF). When shifted to the nonpermissive temperature the cells stop growing and differentiate into macrophage-like cells, as determined by their expression of morphological, functional, and antigenic markers of normal macrophages. They also lose their responsiveness to cMGF and secrete a cMGF-like factor. Ts mutants of E26 retain their leukemogenicity and their ability to transform both erythroblasts and fibroblasts at the nonpermissive temperature, suggesting that the myb oncogene of E26 causes myeloblast transformation and that ets is responsible for erythroblast and fibroblast transformation.

v-mil induces autocrine growth and enhanced tumorigenicity in v-myc-transformed avian macrophages

MH2, an avian retrovirus containing the v-myc and v-mil oncogenes, rapidly transforms chick hematopoietic cells in vitro. The transformed cells belong to the macrophage lineage and proliferate in the absence of exogenous growth factors. Here we analyze a series of MH2 deletion mutants and show that these two oncogenes together establish an autocrine growth system in which v-myc stimulates cell proliferation, while v-mil induces the production of chicken myelomonocytic growth factor (cMGF). We also demonstrate that these two oncogenes cooperate in vivo. MH2 efficiently induces monocytic leukemias and liver tumors, while deletion mutants lacking either a functional v-mil or v-myc do not.

Activation of cMGF expression is a critical step in avian myeloid leukemogenesis

A non‐leukemogenic version of the v‐myb oncogene causes in vitro transformation of avian myeloblasts, which are dependent on chicken myelomonocytic growth factor (cMGF). We have shown that this version of v‐myb, when combined with the erythroleukemia‐inducing v‐erbB oncogene, is capable of causing a mixed myeloid and erythroid leukemia. Myeloid leukemic cells transformed by this construct produce cMGF. To test whether autocrine growth stimulation via cMGF is the essential contribution of the tyrosine kinase oncogene v‐erbB in avian myeloid leukemogenesis we constructed another retrovirus containing both the non‐leukemogenic v‐myb and the cMGF cDNA. This virus induced myeloid leukemia at high efficiency. In a third construct we combined v‐myb with the human EGF‐receptor gene. Myeloid cells transformed by this construct could be stimulated to grow by the addition of cMGF or EGF. Growth stimulation with EGF was blocked by a cMGF antiserum indicating that activation of a normal tyrosine kinase‐type receptor induces cMGF expression but does not bypass the cMGF requirement. We conclude that cMGF plays a key role in the growth regulation of normal and transformed avian myeloid cells.

ts -Oncogene-Transformed Erythroleukemic Cells: A Novel Test System for Purifying and Characterizing Avian Erythroid Growth Factors

An emerging, important characteristic of many leukemic cell types is their altered dependence on and/or response to hematopoietic growth factors [6, 17]. In mammals, many of these growth-regulatory proteins have been purified and the respective genes molecularly cloned [16, 26], but the mechanism by which they regulate growth and differentiation of normal hematopoietic precursors is still poorly understood. This is due partly to the fact that such hematopoietic precursors do not self-renew in vitro and constitute only a minor fraction of bone marrow cells, precluding their purification in large numbers [25]. One particularly successful approach to circumventing this problem was the use of avian retroviral oncogenes that transform hematopoietic precursors [10].

Recombinant Murine Erythropoietin Receptor Expressed in Avian Erythroid Progenitors Mediates Terminal Erythroid Differentiation In Vitro

The biological activity of the recombinant murine erythropoietin receptor (muEpoR) has so far been ascertained only in nonerythroid, established cell lines ectopically expressing the exogenous receptor. Here we show that the regulation of proliferation and differentiation by the muEpoR can be studied in chicken erythroid cells capable of terminal differentiation. The cloned muEpoR was introduced into primary and immortalized chicken erythroblast clones transformed by conditional oncogenes, using retroviral gene transfer. After turning off oncoprotein function, these cells terminally differentiated in response to human erythropoietin (rhu-Epo), similar to cells treated with chicken anemic serum containing avian Epo. Control vector-containing erythroblasts were totally unresponsive to rhu-Epo, but differentiated normally in presence of avian Epo. The avian erythroblasts expressed biologically active muEpoR at physiological levels and bound rhu-Epo with similar high affinity as mammalian erythroblasts expressing endogenous EpoR. Finally, rhu-Epo synergized with insulin in these cells similar to avian Epo. Our results demonstrate that the exogenous muEpoR is able to mediate normal, terminal differentiation in avian erythroid progenitors.

Individual and Combined Effects of Viral Oncogenes in Hematopoietic Cells

Activation of proto-oncogenes is believed to play an important role in the development of human leukemia. For example, chronic myelocytic leukemia is characterized by the presence of a specific translocation which leads to the activation of the c-abl proto-oncogene (see Ben-Neriah and Baltimore, this Vol.). This activation leads to the selective outgrowth of an apparently normal pluripotent stem cell clone and granulocytic cells derived from it. Progeny from this stem cell clone become leukemic in a later step(s) which usually affects myeloid and occasionally also lymphoid cells. Similarly, Burkitt’s lymphoma is associated with a specific translocation which activates the c-myc gene, but this activation is not sufficient to induce the disease (see Klein, this Vol.). In both of these examples the nature of the secondary events required to generate the leukemic state is unclear.