Insights into erythroid differentiation obtained from studies on avian erythroblastosis virus.

Analysis of the oncogenes v-erbB and v-erbA and their normal proto-oncogene counterparts has revealed several novel aspects of erythroid differentiation. A new erythroid progenitor capable of extended self-renewal has been described, tyrosine kinase receptors and steroid hormone receptors have been found to cooperate in controlling self-renewal, and dramatic alterations in the cell cycle have been found to accompany induction of terminal differentiation.

Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum.

Membrane interaction in aggregating cells of Dictyostelium discoideum can be blocked by univalent antibodies directed against specific membrane sites. Using a quantitative technique for measuring cell association, two classes of target sites for blocking antibodies were distinguished and their developmental dynamics studied. One class of these sites is specific for aggregation-competent cells, their quantity rising from virtually 0-level during growth, with a steep increase shortly before cell aggregation. The serological activity of these structures is species specific; they are not detectable in a nonaggregating mutant, but present in a revertant undergoing normal morphogenesis. Patterns of cell assembly in the presence of antibodies show that selective blockage of these membrane sites abolishes the preference for end-to-end association which is typical for aggregating cells. A second class of target sites is present in comparable quantities in particle fractions from both growth-phase and aggregation-competent cells. Blockage of these sites leads to aggregation patterns in which the side-by-side contacts of aggregating cells are abolished. The target sites of aggregation-inhibiting antibodies are suggested to be identical or associated with the molecular units of the cell membrane that mediate cell-to-cell contacts during aggregation. The results indicate that in one cell, two independent classes of contact sites can be simultaneously active.

Epithelial mesenchymal transition by c-Fos estrogen receptor activation involves nuclear translocation of beta-catenin and upregulation of beta-catenin/lymphoid enhancer binding factor-1 transcriptional activity.

Mouse mammary epithelial cells expressing a fusion protein of c-Fos and the estrogen receptor (FosER) formed highly polarized epithelial cell sheets in the absence of estradiol. Beta-catenin and p120(ctn) were exclusively located at the lateral plasma membrane in a tight complex with the adherens junction protein, E-cadherin. Upon activation of FosER by estradiol addition, cells lost epithelial polarity within two days, giving rise to a uniform distribution of junctional proteins along the entire plasma membrane. Most of the beta-catenin and p120(ctn) remained in a complex with E-cadherin at the membrane, but a minor fraction of uncomplexed cytoplasmic beta-catenin increased significantly. The epithelial-mesenchymal cell conversion induced by prolonged estradiol treatment was accompanied by a complete loss of E-cadherin expression, a 70% reduction in beta-catenin protein level, and a change in the expression pattern of p120(ctn) isoforms. In these mesenchymal cells, beta-catenin and p120(ctn) were localized in the cytoplasm and in defined intranuclear structures. Furthermore, beta-catenin colocalized with transcription factor LEF-1 in the nucleus, and coprecipitated with LEF-1-related proteins from cell extracts. Accordingly, beta-catenin- dependent reporter activity was upregulated in mesenchymal cells and could be reduced by transient expression of exogenous E-cadherin. Thus, epithelial mesenchymal conversion in FosER cells may involve beta-catenin signaling.

E-cadherin regulates cell growth by modulating proliferation-dependent beta-catenin transcriptional activity.

beta-Catenin is essential for E-cadherin-mediated cell adhesion in epithelial cells, but it also forms nuclear complexes with high mobility group transcription factors. Using a mouse mammary epithelial cell system, we have shown previously that conversion of epithelial cells to a fibroblastoid phenotype (epithelial-mesenchymal transition) involves downregulation of E-cadherin and upregulation of beta-catenin transcriptional activity. Here, we demonstrate that transient expression of exogenous E-cadherin in both epithelial and fibroblastoid cells arrested cell growth or caused apoptosis, depending on the cellular E-cadherin levels. By expressing E-cadherin subdomains, we show that the growth-suppressive effect of E-cadherin required the presence of its cytoplasmic beta-catenin interaction domain and/or correlated strictly with the ability to negatively interfere with beta-catenin transcriptional activity. Furthermore, coexpression of beta-catenin or lymphoid enhancer binding factor-1 or T cell factor 3 with E-cadherin rescued beta-catenin transcriptional activity and counteracted E-cadherin-mediated cell cycle arrest. Stable expression of E-cadherin in fibroblastoid cells decreased beta-catenin activity and reduced cell growth. Since proliferating cells had a higher beta-catenin activity than G1 phase-arrested or contact-inhibited cells, we conclude that beta-catenin transcriptional activity is essential for cell proliferation and can be controlled by E-cadherin in a cell adhesion-independent manner.

Thyroid hormone receptor/c-erbA: control of commitment and differentiation in the neuronal/chromaffin progenitor line PC12.

The c-erbA proto-oncogenes encode nuclear receptors for thyroid hormone (T3), a hormone intimately involved in mammalian brain maturation. To study thyroid hormone receptor (TR) action on neuronal cells in vitro, we expressed the chicken c-erbA/TR alpha-1 as well as its oncogenic variant v-erbA in the adrenal medulla progenitor cell line PC12. In the absence of T3, exogenous TR alpha-1 inhibits NGF-induced neuronal differentiation and represses neuron-specific gene expression. In contrast, TR alpha-1 allows normal differentiation and neuronal gene expression to occur in the presence of T3. Finally, TR alpha-1-expressing cells become NGF-responsive for proliferation when T3 is absent, but NGF-dependent for survival in presence of T3. A similar differentiation induction by NGF plus T3 was observed in a central nervous system-derived neuronal cell line (E 18) expressing exogenous TR alpha-1. Together with the finding that TR alpha-1 constitutively blocked dexamethasone-induced differentiation of PC12 cells into the chromaffin pathway, these results suggest that TR alpha-1 plays an important role in regulating commitment and maturation of neuronal progenitors. In contrast, the v-erbA oncogene, a mutated, oncogenic version of TR alpha-1, partially but constitutively inhibited NGF-induced neuronal differentiation of PC12 cells and potentiated dexamethasone-induced chromaffin differentiation, giving rise to an aberrant "interlineage" cell phenotype.

Mammalian granulocyte-macrophage colony-stimulating factor receptor expressed in primary avian hematopoietic progenitors: lineage-specific regulation of proliferation and differentiation.

The cytokine Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) regulates proliferation, differentiation, and apoptosis during myelopoiesis and erythropoiesis. Structure-function relationships of GM-CSF interactions with its receptor (GM-R), the biochemistry of GM-R signal transduction, and GM-CSF action in vivo are relatively well understood. Much less is known, however, about GM-R function in primary hematopoietic cells. In this paper we show that expression of the human GM-R in a heterologous cell system (primary avian erythroid and myeloid cells) confirms respective results in murine or human cell lines, but also provides new insights how the GM-R regulates progenitor proliferation and differentiation. As expected, the hGM-CSF stimulated myeloid progenitor proliferation and differentiation and enhanced erythroid progenitor proliferation during terminal differentiation. In the latter cells, however, the hGM-R only partially substituted for the activities of the erythropoietin receptor (EpoR). It failed to replace the EpoR in its cooperation with c-Kit to induce long-term proliferation of erythroid progenitors. Furthermore, the hGM-R alpha chain specifically interfered with EpoR signaling, an activity neither seen for the betac subunit of the receptor complex alone, nor for the alpha chain of the closely related Interleukin-3 receptor. These results point to a novel role of the GM-R alpha chain in defining cell type-specific functions of the GM-R.

The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions.

Members of the epidermal growth factor (EGF) receptor family are known to be specifically involved in mammary carcinogenesis. As a nuclear target of activated receptors, we examined c-Jun in mammary epithelial cells. For this, we used a c-JunER fusion protein which was tightly controlled by estrogen. Activation of the JunER by hormone resulted in the transcriptional regulation of a variety of AP-1 target genes. Hormone-activated JunER induced the loss of epithelial polarity, a disruption of intercellular junctions and normal barrier function and the formation of irregular multilayers. These changes were completely reversible upon hormone withdrawal. Loss of epithelial polarity involved redistribution of both apical and basolateral proteins to the entire plasma membrane. The redistribution of E-cadherin and beta-catenin was accompanied by a destabilization of complexes formed between these two proteins, leading to an enrichment of beta-catenin in the detergent-soluble fraction. Uninduced cells were able to form three-dimensional tubular structures in collagen I gels which were disrupted upon JunER activation, leading to irregular cell aggregates. The JunER-induced disruption of tubular structures was dependent on active signaling by growth factors. Moreover, the effects of JunER could be mimicked in normal cells by the addition of acidic fibroblast growth factor (aFGF). These data suggest that a possible function of c-Jun in epithelial cells is to modulate epithelial polarity and regulate tissue organization, processes which may be equally important for both normal breast development and as initiating steps in carcinogenesis.

Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton.

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.

Cell dissociation: univalent antibodies as a possible alternative to proteolytic enzymes.

Univalent antibody fragments directed against special membrane antigens dissociate multicellular bodies of the cellular slime mold Dictyostelium discoideum completely into single cells. This provides a gentle method for cell dissociation and demonstrates that a nonenzyme protein can disintegrate a tissue

Translation control: bridging the gap between genomics and proteomics?

mRNA profiling enables the expression levels of thousands of transcripts in a cell to be monitored simultaneously. Nevertheless, analyses in yeast and mammalian cells have demonstrated that mRNA levels alone are unreliable indicators of the corresponding protein abundances. This discrepancy between mRNA and protein levels argues for the relevance of additional control mechanisms besides transcription. As translational control is a major mechanism regulating gene expression, the use of translated mRNA in profiling experiments might depict the proteome more closely than does the use of total mRNA. This would combine the technical potential of genomics with the physiological relevance of proteomics.

PDGF essentially links TGF-beta signaling to nuclear beta-catenin accumulation in hepatocellular carcinoma progression.

The cooperation of Ras - extracellular signal-regulated kinase/mitogen-activated protein kinase and transforming growth factor (TGF)-beta signaling provokes an epithelial to mesenchymal transition (EMT) of differentiated p19(ARF) null hepatocytes, which is accompanied by a shift in malignancy and gain of metastatic properties. Upon EMT, TGF-beta induces the secretion and autocrine regulation of platelet-derived growth factor (PDGF) by upregulation of PDGF-A and both PDGF receptors. Here, we demonstrate by loss-of-function analyses that PDGF provides adhesive and migratory properties in vitro as well as proliferative stimuli during tumor formation. PDGF signaling resulted in the activation of phosphatidylinositol-3 kinase, and furthermore associated with nuclear beta-catenin accumulation upon EMT. Hepatocytes expressing constitutively active beta-catenin or its negative regulator Axin were employed to study the impact of nuclear beta-catenin. Unexpectedly, active beta-catenin failed to accelerate proliferation during tumor formation, but in contrast, correlated with growth arrest. Nuclear localization of beta-catenin was accompanied by strong expression of the Cdk inhibitor p16(INK4A) and the concomitant induction of the beta-catenin target genes cyclin D1 and c-myc. In addition, active beta-catenin revealed protection of malignant hepatocytes against anoikis, which provides a prerequisite for the dissemination of carcinoma. From these data, we conclude that TGF-beta acts tumor progressive by induction of PDGF signaling and subsequent activation of beta-catenin, which endows a subpopulation of neoplastic hepatocytes with features of cancer stem cells..

The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity.

Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, we identified transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell-cell adhesion, including the cell polarity genes Crumbs3, HUGL2 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promotor activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour-host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. Our data show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.

Raf plus TGFbeta-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin.

Oncogenic Ras interferes with adhesive functions of epithelial cells, but requires tumor growth factor beta (TGFbeta) signaling to cause epithelial-mesenchymal transition (EMT) and tumor progression in model systems. To investigate the mechanisms by which Ras and TGFbeta pathways cooperate in EMT induction, we introduced a tamoxifen-inducible version of Raf-1 (RafER) into fully polarized, mammary epithelial cells (EpH4). EMT characterized by loss of E-cadherin expression and upregulation of invasiveness-promoting genes was induced by TGFbeta plus 4-hydroxytamoxifen (4HT) activation of RafER. Downregulation of E-cadherin by RafER plus TGFbeta was detectable in total cell lysates after 48 h and much earlier in detergent-insoluble fractions of E-cadherin. Both pathways cooperated to strongly enhance endocytosis of E-cadherin, mainly via the clathrin-dependent route. Pulse-chase experiments showed decreased E-cadherin protein stability in cells stimulated with TGFbeta and 4HT and increased E-cadherin half-life in the presence of monensin. Monensin and chloroquine prevented E-cadherin degradation to different extent, but only monensin effectively blocked the loss of E-cadherin from the junctional complexes. Both lysosome inhibitors caused accumulation of E-cadherin vesicles, some of which were positive for Cathepsin D and lysosome-associated membrane protein 1 (LAMP-1). In addition, TGFbeta and mitogen-activated protein kinase hyperactivation synergistically induced E-cadherin ubiquitination, suggesting that the cooperation of Raf and TGFbeta favors lysosomal degradation of E-cadherin instead of its recycling. Our data indicate that early stages of EMT involve cooperative, post-translational downregulation of E-cadherin, whereas loss of E-cadherin via transcriptional repression is a late event in EMT.

Erythroid progenitor renewal versus differentiation: genetic evidence for cell autonomous, essential functions of EpoR, Stat5 and the GR.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

A crucial function of PDGF in TGF-beta-mediated cancer progression of hepatocytes.

Polarized hepatocytes expressing hyperactive Ha-Ras adopt an invasive and metastatic phenotype in cooperation with transforming growth factor (TGF)-beta. This dramatic increase in malignancy is displayed by an epithelial to mesenchymal transition (EMT), which mimics the TGF-beta-mediated progression of human hepatocellular carcinomas. In culture, hepatocellular EMT occurs highly synchronously, facilitating the analysis of molecular events underlying the various stages of this process. Here, we show that in response to TGF-beta, phosphorylated Smads rapidly translocated into the nucleus and activated transcription of target genes such as E-cadherin repressors of the Snail superfamily, causing loss of cell adhesion. Within the TGF-beta superfamily of cytokines, TGF-beta1, -beta2 and -beta3 were specific for the induction of hepatocellular EMT. Expression profiling of EMT kinetics revealed 78 up- and 235 downregulated genes, which preferentially modulate metabolic activities, extracellular matrix composition, transcriptional activities and cell survival. Independent of the genetic background, platelet-derived growth factor (PDGF)-A ligand and both PDGF receptor subunits were highly elevated, together with autocrine secretion of bioactive PDGF. Interference with PDGF signalling by employing hepatocytes expressing the dominant-negative PDGF-alpha receptor revealed decreased TGF-beta-induced migration in vitro and efficient suppression of tumour growth in vivo. In conclusion, these results provide evidence for a crucial role of PDGF in TGF-beta-mediated tumour progression of hepatocytes and suggest PDGF as a target for therapeutic intervention in liver cancer.

TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis.

BACKGROUND: Invasive growth of epithelial tumor cells, a major cause of death from cancer in humans, involves loss of epithelial polarity and dedifferentiation. Transforming growth factor beta (TGFbeta) is regarded as a major tumor suppressor during early tumor development because it inhibits cell-cycle progression and tumor growth. Many dedifferentiated, late-stage tumors are resistant to growth inhibition by TGFbeta, however, and even secrete TGFbeta. In line with this, TGFbeta is involved in angiogenesis, wound healing and epithelial-mesenchymal transition (EMT) during development. Ha-Ras-transformed mammary epithelial cells (EpRas) undergo TGFbeta-induced EMT maintained via a TGFbeta autocrine loop. Thus, we have analyzed whether signal transduction by the TGFbeta receptor (TGFbetaR) is required for local tumor cell invasion and metastasis. RESULTS: A dominant-negative type II TGFbetaR (TGFbetaRII-dn) was expressed using retroviral vectors in EpRas cells and highly metastatic mesenchymal mouse colon carcinoma cells (CT26). In both cell types, TGFbetaRII-dn blocked TGFbetaR signaling and heavily delayed tumor formation. In EpRas cells, TGFbetaRII-dn prevented EMT. In the dedifferentiated mesenchymal CT26 cells, TGFbetaRII-dn caused mesenchymal-to-epithelial transition and inhibited their in vitro invasiveness in several assays. In addition, TGFbetaRII-dn completely abolished metastasis formation by CT26 cells. Furthermore, several human carcinoma lines lost in vitro invasiveness when treated with neutralizing TGFbeta antibodies or soluble receptor variants. Finally, human colon carcinoma cells (hnPCC) expressing a mutated, non-functional TGFbetaRII were non-invasive in vitro, a defect restored by re-expressing wild-type TGFbetaRII. CONCLUSIONS: Cell-autonomous TGFbeta signaling is required for both induction and maintenance of in vitro invasiveness and metastasis during late-stage tumorigenesis. TGFbetaRII therefore represents a potential target for therapeutical intervention in human tumorigenesis.

Primary, self-renewing erythroid progenitors develop through activation of both tyrosine kinase and steroid hormone receptors.

BACKGROUND: Self renewal in the hematopoietic system is thought to be restricted to a class of pluripotent stem cells. The capacity of cells with the properties of committed progenitors to self renew in many leukemias is thought to be an abnormal property resulting from the mutations responsible for leukemic transformation. It is not known how cells that can self-renew differ from cells that cannot. The notion that only pluripotent stem cells self renew has recently been challenged: normal committed erythroid progenitors capable of sustained self renewal have been described. These cells, called SCF/TGF alpha progenitors, co-express the c-Kit receptor tyrosine kinase and c-ErbB, the avian receptor for epidermal growth factor and transforming growth factor (TGF) alpha, and they undergo continuous self renewal in response to TGF alpha and estradiol. In contrast, common erythroid progenitors (termed SCF progenitors) express only c-Kit and undergo a limited number of cell divisions in response to the c-Kit ligand, stem cell factor (SCF). Both types of progenitor faithfully reproduce terminal erythroid differentiation in vitro when exposed to differentiation factors. Here, we have investigated the developmental origin of these two classes of self-renewing erythroid progenitors. RESULTS: We show that SCF progenitors can develop into SCF/TGF alpha progenitors. This developmental conversion requires 10-14 days and is accompanied by a gradual up-regulation of bioactive TGF alpha receptor. Using sera depleted of endogenous growth factors, we demonstrate that the development of SCF progenitors into SCF/TGF alpha progenitors absolutely requires the simultaneous presence of SCF, TGF alpha and estradiol, and is strongly enhanced by an unknown activity in chicken serum. CONCLUSIONS: SCF progenitors can be induced to develop into self-renewing SCF/TGF alpha progenitors. The development of self renewal is triggered by specific combinations of growth factors and hormones. This has important implications for understanding leukemogenesis, as the self renewal of leukemic cells may reflect the normal potential of certain committed progenitor cells and not, as has been thought, a unique abnormal property of leukemic cells.

A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.

Oncornavirus-induced sarcoma formation obscured by rapid development of lethal leukemia.

Injection i.v. of avian erythroblastosis virus (AEV) strain ES4 causes a high incidence of leukemia and the death of most of the inoculated chicks within 2 weeks. As found earlier, the virus is defective for replication and transforms bone marrow cultures in vitro, and surprisingly, also chick embryo fibroblasts. Inoculation of transformed AEV cells negative for virus production into newborn chicks induced the formation of sarcomas only, whereas cells superinfected with helper virus induced the formation of erythroblastosis in addition to sarcomas. The helper virus alone caused neither sarcomas nor erythroblastosis during the experimental period. These findings were explained by the hypothesis that AEV-induced erythroblastosis develops more rapidly than do AEV-induced sarcomas and that animals receiving i.v. injections die of the leukemia before sarcomas become detectable. The observation that animals receiving i.m. injections of AEV developed sarcomas at the site of injection strongly supports this concept. Most of the animals that received i.m. injections also developed an erythroblastosis that was delayed, however, in comparison to the animals receiving i.v. injections. Our data also suggest that the erythroblastosis induced by AEV does not suppress the formation of sarcomas in the same animal.

The v-erbA oncogene requires cooperation with tyrosine kinases to arrest erythroid differentiation induced by ligand-activated endogenous c-erbA and retinoic acid receptor.

The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), cooperates with tyrosine kinase oncogenes in erythroblast transformation. Here we show that the ligand-activated, endogenous retinoic acid receptor (RAR-alpha), in cooperation with c-erbA/TR-alpha, efficiently reverses the transforming effect of kinase oncogenes, overcoming oncogene-induced self-renewal by triggering terminal differentiation of the transformed cells into healthy erythrocytes. This differentiation induction was accompanied by up-regulation of erythrocyte gene expression. Similarly, RAR-alpha and over-expressed exogenous c-erbA/TR-alpha efficiently abolished the differentiation arrest caused by v-erbA, while the low levels of endogenous TR-alpha had no effect. In contrast, transformation by v-erbA plus a kinase oncogene was not affected at all by ligand-activated endogenous or over-expressed exogenous TR-alpha and RAR-alpha. These results suggest that oncogene cooperation is required to protect leukemic erythroblasts from differentiation induction via endogenous, nuclear hormone receptors. Endogenous c-erbA/TR-alpha and RAR-alpha apparently cooperated in abolishing erythroblast self-renewal and inducing differentiation, since the respective ligands acted in a synergistic fashion, and overexpressed, non-ligand-bound c-erbA/TR-alpha suppressed endogenous RAR-alpha function in differentiation induction. Genetic evidence is presented that this functional cooperation requires the receptor dimerization domain, suggesting that TR-alpha/RAR-alpha heterodimers play a role in regulation of erythroid differentiation.