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The expression of the retinoblastoma susceptibility (RB-1) gene was investigated in highly proliferating mouse embryonic stem (ES) cells and in slowly proliferating mouse embryonic fibroblasts. The RB protein was expressed at the same level in these two cell types. Mainly hyperphosphorylated RB was detected in exponentially-growing ES cells. Embryonic fibroblasts and embryonic stem cells were synchronized by colcemid block followed by mitotic shake-off. In embryonic fibroblasts, DNA replication started 10-15 h after exit from mitosis and RB was transiently dephosphorylated during the G1 phase as previously described. In ES cells, DNA replication started 2 h after release from the colcemid block but virtually no hypophosphorylated RB was observed after the release. Instead, there was a dramatic decrease in the total RB protein level between exit from mitosis and entry into S phase. These observations were made by using two different monoclonal antibodies, both in immunoblotting and immunoprecipitation experiments. Absence of hypophosphorylated RB and cell cycle-dependent change in total RB protein level may be relevant to the high proliferation rate and to the tumorigenic nature of mouse embryonic stem cells.

In AEV-transformed erythroleukemic cells the v-erbA gene product is likely to antagonize the function of triiodothyronine (T3) and retinoic acid (RA) receptors and thereby to block cell differentiation. We have thus investigated the effects of T3 and RA on normal early erythrocytic progenitor cells. Here we show: (1) that either RA or T3 play an essential role during the early commitment to erythrocytic differentiation, (2) that both T3 and RA induce death by apoptosis and a strong inhibition of self-renewal in progenitor cells grown in the absence of differentiation-inducing agents and (3) that the v-erbA oncogene renders erythrocytic progenitor cells insensitive to apoptosis and to self-renewal inhibition induced by RA or T3. The behaviour of a non-transforming mutant of v-erbA suggests that this v-erbA-induced protection is related to its transforming potential.

Transforming growth factor beta 1 (TGF-beta 1) inhibits growth of primary cultures of chick embryo fibroblasts by affecting G1 and strongly increasing the generation time. This inhibition is reversed by the nuclear oncogenes v-jun, v-fos, v-myc, but not v-erbA and v-ets. It is also reversed by v-myb from either avian myeloblastosis virus or avian E26 retrovirus. Taken together, these results strongly suggest that independent, functional interferences may take place between the TGF-beta 1-induced growth inhibitory pathway and the oncogen-driven stimulatory pathway(s) at the level of the AP-1, Myc, and Myb transcription factors.

The human BTG1 gene (expressing an anti-proliferative function) is an evolutionarily conserved gene homologous to the murine PC3/TIS21 genes. Here, we report the cloning and sequencing of the murine BTG1 coding region and chicken BTG1 cDNA. The putative human and mouse BTG1 proteins are 100% identical; the chicken BTG1 cDNA contains an open reading frame of 170 amino acids with a 91% identity to its human and murine counterparts. The 3'-untranslated region of BTG1 is also highly conserved (82% homology between human and chicken), suggesting that it plays a key role in the regulation of BTG1 expression. These data confirm that BTG1 is phylogenetically highly conserved and that BTG1 and PC3/TIS21 may constitute the first members of a new family of functionally related genes.

The avian myeloblastosis virus (AMV) causes monoblastic leukemia in the chick. Two non-producer clones of AMV-transformed monoblasts, BM2/C3A and BM2L/A2B5, have been described (see Bottazzi et al., this issue). They differ in their growth requirements and in their ability to induce leukemia when injected into the chick embryo. We first genetically tagged these clones by retroviral infection with a vector expressing the bacterial lacZ gene. Then, we injected the lacZ-positive cells via the chorioallantoic vein into chick embryos. With BM2L/A2B5 cells, the bone marrow of the injected birds was rapidly invaded by lacZ-positive cells. In addition, these cells rapidly overgrew cultures of bone marrow cells derived from injected animals. Conversely, the growth of BM2/C3A was inhibited in the injected animals and only a few blue cells, with the morphology of macrophages, were detected in cultures of bone marrow cells. We developed an in vitro assay to mimic in vitro the differential growth of BM2/C3A and BM2L/A2B5 observed in vivo. These data strongly suggest that BM2/C3A cells retain their ability to differentiate into macrophages in the normal bone marrow environment and that BM2L/A2B5 cells differ from BMC/C3A in the loss of this capacity.

Quail cells were immortalized for the first time by using retroviruses expressing the 12S adenoviral E1A gene. In these cells, interaction between the 12S E1A product and the quail RB protein was shown, suggesting that the 12S adenoviral E1A product works in avian cells through similar biochemical pathways as in mammalian cells by interacting and inactivating host cellular proteins, including the RB product. These results confirm that the RB product exhibits a universal function among higher vertebrates in controlling cellular growth and tumor progression.

We have studied the transforming ability of the oncogenes erbA and/or erbB in chicken neuroretina (CNR) cells and the effect of basic fibroblast growth factor (bFGF) on the transformed phenotype. The erbA oncogene alone was only transforming in the presence of bFGF. In contrast, cells expressing erbB as well as erbA + erbB were transformed in a bFGF-independent manner and were unresponsive to the growth factor. We studied whether other oncogenes could also block the cooperation between erbA and bFGF. Cytoplasmic or membrane-bound oncogenes (src, ras, or mill raf) increased the transforming potential of erbA but rendered the cells unresponsive to bFGF. Conversely, the nuclear oncogenes tested (fos and myb-ets + myc) also cooperated with erbA in CNR cell transformation but the cells remained responsive to the growth factor. A likely explanation is that CNR cells carrying the cytoplasmic but not the nuclear oncogenes have already activated the bFGF signal transduction pathway.

The genome of the avian leukemia virus E26 is a unique example of association between two transcription factors which appear as a fused composite nuclear oncoprotein, P135gag-myb-ets. Previous studies with E26 have shown that v-myb and v-ets must cooperate to fully transform both erythrocytic and myelomonocytic precursor cells in vivo and in vitro. To analyse further the contribution of the individual domains involved in the transformation of various hematopoietic lineages, we have constructed several mutant viruses expressing a fusion protein with deletions in either v-myb or v-ets. We show here that integrity of the v-ets oncogene is necessary for transformation of the erythrocytic cells but that neither the DNA-binding domain nor the trans-activating domain of v-myb is required for this transformation. The DNA-binding domain of v-ets is necessary to transform myelomonocytic cells. Furthermore, we show that E26 onco-protein also transforms granulocytic cells. The v-ets DNA-binding domain is not necessary to transform them, whereas deleting the v-myb DNA-binding domain strongly reduces transformation of these cells. These data show that the v-myb and v-ets DNA-binding domains provide quite different contributions to the transformation of various hematopoietic lineages by E26.

The effect of the v-erbA and/or v-erbB oncogenes on cellular gene expression was investigated after separation by two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labelled proteins from chicken embryo fibroblasts (CEF), infected by either the avian erythroblastosis virus (AEV) carrying both oncogenes, or by viruses carrying only one of them. We observed significant changes in the synthesis of 34 proteins in AEV-transformed CEF as compared with control cells. The synthesis of 24 of them was increased while the synthesis of the other 10 proteins was decreased. The expression of v-erbB alone is necessary and sufficient to induce changes in the synthesis of 27 proteins while the 7 remaining modifications are observed only in cells expressing v-erbB together with v-erbA. Moreover, the deregulation of protein synthesis by v-erbB-expressing viruses was correlated with the morphological transformation state of cells.

Ligand-activated retinoic acid receptor alpha (RAR alpha) and c-ErbA alpha repress the AP-1-mediated transcriptional activation of the interstitial collagenase gene promoter by specifically decreasing the activity of the AP-1 transcription factor. On the other hand, the v-ErbA oncoprotein fails to repress the AP-1 activity and acts as a dominant negative oncoprotein by overcoming the repression of the AP-1 activity induced by RAR alpha and c-ErbA alpha. This maintenance by v-ErbA of a fully active AP-1 complex is correlated with the abrogation by this same oncogene product of the growth-inhibitory response of chicken embryo fibroblasts to retinoic acid treatment. This new mechanism of action of v-ErbA together with its previously discovered dominant repressor effect on transcription of thyroid hormone-activated target genes may explain the contribution of the v-erbA oncogene to sarcomatogenic and leukemogenic transformation.

Retinoic acid inhibits chicken embryo fibroblast (CEF) proliferation by altering the G1 phase of the cell cycle with induction of a strong increase in the generation time. This growth-inhibitory response to retinoic acid is abrogated by expression of the v-erbA oncogene, suggesting an interference between retinoic acid receptors and the v-ErbA oncoprotein. Moreover, CEF expressing either the v-src, v-jun or v-fos oncogenes are also insensitive to retinoic acid treatment. In contrast, CEF expressing either the v-myc, v-myb-ets, v-mil, v-sea or v-erbB oncogenes are still sensitive to retinoic acid. These data strongly suggest functional interferences between the retinoic acid receptors and the AP-1 transcription factor complex in the control of expression of genes involved in CEF proliferation.

We performed molecular cloning and sequencing of the breakpoints of a new chromosomal translocation involving the MYC protooncogene locus. This secondary t(8;12)(q24;q22) was associated with a primary t(11;14)(q13;q32) translocation in a case of B-cell chronic lymphocytic leukemia (CLL) in blastic transformation. In this leukemia, Northern blot and nuclease analyses SI showed that MYC was strongly expressed with initiation of the transcription at both the 5' and 3' promoters as observed in Burkitt's lymphomas; no coding change was observed in MYC putative regulatory sequences. The breakpoint on chromosome 8 mapped to the 3' end of the MYC locus, in a region containing a potential Z-DNA tract, and where we identified two DNase 1 hypersensitive sites. A rearranged MYC gene fragment was cloned and shown to contain chromosome 12 information by Southern blot analysis and by in situ hybridization. A genomic probe subcloned from the isolated region of the chromosome 12 recognized a 1.8 kb transcript in virtually all the tissues tested but a preferential expression of this new gene, which we termed BTG1 (for B-cell translocation gene 1) was observed in the CLL cells and in tissues of lymphoid origin. This chromosome 12 coding sequence is conserved in evolution and a transcript of similar size is present in murine tissues.

We report that the activation of the endogenous chicken EGF receptor leads to the tumorigenic growth in vivo of early passage chicken embryo fibroblasts (CEFs) that express a nonsarcomagenic oncogene, v-myc. To provide a continuous paracrine source of this growth factor in vivo, we employed irradiated Rat-1 cells which had been stably transfected with a synthetic cDNA to human EGF. Expression of another non-sarcomagenic nuclear oncogene, v-erbA, prones the CEFs to in vitro transformation by EGF, but does not cause EGF dependent tumorigenicity in vivo. The short period of incubation in the in vivo assay employed by our study (10 days), together with the genetic stability of primary chicken embryo fibroblasts, make it very likely that the reported alterations in cellular behaviour are a direct and primary effect of the expression of the relevant oncogenes and their cooperation with the EGF induced response. Dose response and ligand binding assays suggest that the EGF response is transmitted via the chicken c-erbB molecule, which by virtue of its preference for TGF-alfa is distinct from the mammalian EGF receptors studied so far. The level of expression of the endogenous chicken EGF receptor is within the same range as that reported for primary human fibroblasts (5-7 x 10(3) per cell). The cooperative effect of v-myc with chicken c-erbB probably takes place at a post receptor level, as its expression did not affect the steady state level or affinity for ligand of the chicken EGF receptor.

The identification of the viral oncogene v-erbA carried by an avian leukemia retrovirus has directly demonstrated the involvement of hormone receptors in neoplastic transformation. v-erbA represents an altered form of a nuclear receptor of the thyroid hormone T3. It blocks the differentiation of chicken erythrocyte progenitor cells and contributes to sarcoma transformation in association with other oncogenes. The protein encoded by v-erbA behaves as an antagonist against the normal T3 receptors and retinoic acid receptors. The primary effects of the protein result in altering the transcription of genes normally under control of the intact receptors. Presumably among these target genes are to be found genes which control cell differentiation and proliferation.

The coding sequences of avian (quail) or murine c-jun proto-oncogenes were introduced into a non-defective retroviral vector derived from Rous sarcoma virus (RSV) in which c-jun replaces v-src. Primary avian fibroblasts chronically infected with either one of these viruses exhibit some phenotypic traits characteristic of RSV-transformed cells, including sustained growth in low serum medium and ability to develop colonies from single cells in agar, even though they are still of normal morphology and contact inhibited. This altered growth control correlates with enhanced AP1-specific DNA binding activity as well as with higher levels of c-Jun products. Unexpectedly, repression of the endogenous c-Jun product is observed in cells overexpressing murine c-Jun. Cells expressing the avian and the murine c-Jun products display qualitatively similar phenotypes; nevertheless, for every transformed trait considered, the murine c-jun seemed more potent than its quail homologue. These data suggest that the avian or murine c-jun proto-oncogenes may trigger a subset of the 'transforming functions' normally induced by v-src, and which are more specifically related to growth in low serum and in the absence of solid support.

The v-erbA oncogene encodes an altered form of the nuclear receptor of the thyroid hormone triiodothyronine (T3). This altered receptor is unable to bind T3, and blocks the differentiation of chicken erythrocyte progenitor cells. To identify the cellular target genes of v-ErbA in the transformed cells, we analysed the expression of several genes in normal erythrocytic cells exposed to T3, and found that the gene encoding carbonic anhydrase II is transcriptionally activated by the hormone. In contrast, this gene is repressed in erythroleukemic cells transformed by the v-erbA product. To investigate in more details the effects of v-ErbA, we constructed a mutant of v-ErbA in which we restored the ability to bind T3. This mutant developed its oncogenicity only in the absence of T3. Upon binding of T3, the transformed cells differentiated and immediately expressed the carbonic anhydrase II gene. These data show that v-ErbA directly inhibits the transcription of the carbonic anhydrase II gene, presumably by competing with normal T3 receptors. The carbonic anhydrase II gene is the first identified target gene of the v-ErbA oncoprotein in erythroleukemic cells.

Oncogenic cell transformation induces major changes in the structure and physiology of the cells: modifications of morphology, differentiation block, disorganisation of cytoskeleton and extracellular matrix, alterations in growth control. The identification of oncogenes relies upon transfer into host normal cells of DNA isolated from cancer cells. The recent development of DNA transfer into germinal cells has provided new insights into the genetic control of tumorogenesis in vivo. In most cases, full transformation into leukemic or tumor cell requires the cooperation of several oncogenes. These observations support the hypothesis of cancer as a multistep process. However, many of the cooperative oncogenes have not yet been identified, especially in human cancers. The recent discovery of genes acting as repressors of cell growth in normal cells has brought to light a new class of potential recessive oncogenes that might have a contributory function in cancer development.

To determine the function of c-fps in chicken macrophages and granulocytic cells we have infected chicken bone marrow cells with retroviruses containing the v-fps oncogene. Normal chicken macrophage progenitors, M-CFCs, give rise to macrophage colonies in semisolid cultures when macrophage colony stimulating factor (M-CSF) is added into the culture medium. Upon infection with v-fps bearing retroviruses, we observed that M-CFCs were induced to develop macrophage colonies in vitro without exogenous M-CSF. This activation results from a direct effect of v-fps on the M-CFCs. No leukemic transformation was observed in the infected colonies. By comparing the effects of several retroviruses, we showed that the induction of M-CFC development is specific to v-fps containing viruses and mediated by the v-fps protein. These observations support the hypothesis that the c-fps gene is involved in the control of proliferation and/or differentiation of myeloid cells.

Haemopoietic stem cells from donors of different ages (embryos, adults) have been grafted into irradiated hosts. Cell multiplication kinetics, foetal haemoglobin production, age-specific antigen production were examined during two weeks after grafting. The results obtained tend to show that the erythrocyte characteristics are, for a large part, already determined in a stem cell type endowed with a large proliferative capacity.

A factor present in normal mouse serum stimulates proliferation of late erythroid precursors grown in cultures in vitro. This factor was shown not to have a corrective effect on culture conditions by the following criteria: (a) CFU-E frequency in the absence of NMS was at least as great as published data for various mouse strains, (b) an inhibitory effect of endotoxin was ruled out, (c) sensitivity of erythroid precursors to erythropoietin was similar in the presence or absence of NMS, and (d) the number of colonies was linearly related to the cell dose. The enhancing effect of NMS was independent of hemin, transferrin, or dexamethasone, products all known to be stimulators of erythropoiesis. It was shown to be specific for CFU-E. We propose that this material be termed erythropoietic stimulating cofactor (ESCF).

The authors report the case of a boy with a history of recurrent infections, who presented with erythroblastopenia at age 5 years. An immune deficiency with hyper IgM was found. Erythroblastopenia appeared to be related to a serum inhibitor of erythropoiesis. This inhibitor was present in the serum IgM fraction and acts by blocking the erythropoietic activity of a soluble substance in the serum, which is not erythropoietin but which could be a cofactor of this hormone. A series of 5 plasma exchanges, performed in 10 days, permitted to clear the inhibitor and induced remission, still persisting after a 12 month follow-up.

The enumeration of erythropoietic colony-forming cells in vitro has allowed us to complete previous data on changes in the various erythroid cell populations during chick embryo-genesis. Erythrocytic colony-forming units in culture (CFU-cE) which are sensitive to avian erythropoietin appear in the blastoderm as soon as the 24th hour of development. They represent most likely precursors of the megalocytic erythropoiesis, and do not seem to derive from stem cells common with normocytic erythropoiesis. Data concerning vitelline normocytic erythropoiesis were analysed in a kinetic model based on stochastic change of the stem cells. From this model it appears that 17-20 cell divisions are required for differentiation of erythrocytes from stem cells.

Irradiated chicken are injected with haemopoietic tissues from adult or 11-day-old embryos. Development of stem cells gives rise to well-defined erythrocytic colonies on the surface of the tibial marrow. Erythropoietic production appears to be similar from adult marrow and embryonic blood stem cells; production from injected vitelline stem cells seems to be between 3 and 4 times higher than that of adult marrow stem cells. Results are discussed on the basis of two hypotheses: -existence of an extramedullary erythropoietic site in the host after vitelline cells grafting; -development of vitelline stem cells in the host marrow with kinetic patterns different from those of grafted adult marrow or embryonic blood stem cells. Anyway, the 11-day-old embryo appears to contain at least two types of blood stem cells with distinctive properties. Developmental origin, relationship and future of these different stem cells remain to be analysed.

The dual leukemogenic response, involving both the erythroid and myeloid hemopoietic systems in chickens infected with E26 virus, has previously been described (C. Moscovici, J. Samarut, L. Gazzolo, and M. G. Moscovici, 1981. Virology 113, 765-768; K. Radke, H. Beug, S. Kornfeld, and T. Graf, 1982. Cell 31, 643-653). Similarly, the in vitro response of the two lineages resulted in the concomitant transformation and proliferation of erythroblast and myeloblast leukemic cells. The present study, using embryonic tissues at very early stages of development, was valuable in implying that E26 target cells are recruited among uncommitted erythroid-myeloid stem cells as well as myeloid- or erythroid-committed progenitor cells. Therefore, E26 may be the first avian retrovirus capable of interacting with uncommitted hemopoietic precursor cells.

The production of erythrocytes bearing an "immature" antigen (Im+ cells) and a "fetal" antigen (Ft+ cells) has been studied in irradiated adult mice grafted either with fetal liver or adult bone marrow cells. The Im+ cells reach a peak 8-11 days after grafting. Ft+ cells are detected only after graft of fetal liver cells; the younger the liver, the greater the number. Since Ft+ cells are rapidly and briefly produced, they could be the progeny of erythroid-committed precursors, which are particularly numerous among fetal liver cells. Environmental factors directing the erythropoietic differentiation towards Ft+ erythrocytes in fetuses or Ft- erythrocytes in adults are proposed.

Avian erythroblastosis virus (AEV-ES4), a transforming avian retrovirus, transforms chicken embryo fibroblasts (CEFs) in culture and induces the maintenance of ribosomal protein S6 phosphorylation in the absence of serum. This effect is less pronounced after AEV-ES4 transformation than after transformation by Rous sarcoma virus (PR-RSV A). However, our results indicate that the two viruses induce an activation of the same S6 phosphokinase, as evidenced by the identity of S6 phosphopeptides and phosphoaminoacids in the two cases. Moreover this activation is performed through a protein kinase C-independent pathway. Expression of the v-erbA oncogene alone, which enhances the growth potential of CEFs, is not able to maintain S6 phosphorylation either in the absence of serum or in the presence of low serum concentration (0.5%). Expression of the v-erbB oncogene alone is responsible for all these AEV-ES4-induced effects. Furthermore, the maintenance of S6 phosphorylation in the absence of serum might be correlated with the degree of transformation of AEV-ES4-infected CEFs. These results show that S6 phosphorylation is one of the biochemical mechanisms deregulated by v-erbB expression and is involved in the transformation process.

A culture method is proposed for the in vitro development of chicken erythrocytic progenitors. When grown with avian erythropoietin, Colony Forming Unit Erythrocytic (CFU-E) and Burst Forming Unit-Erythrocytic (BFU-E) give rise respectively to erythrocytic colonies and bursts within 3 and 6 days. BFU-E development is greatly enhanced by pokeweed-mitogen-spleen-cell conditioned medium and requires higher erythropoietin concentrations than for CFU-E. An antigen specific to immature red cells can be detected on CFU-E but not on BFU-E, showing that both progenitors represent distinct entities. BFU-E and CFU-E are found in embryonic marrow and yolk sac. In the young blastoderm BFU-E becomes detectable at the primitive streak stage.

In contrast to uninfected chicken embryo fibroblasts (CEFs), CEFs infected with a retroviral vector that carries the v-erbA gene of avian erythroblastosis virus displayed new properties. These included limited anchorage-independent growth in soft agar, growth without latency in serum-supplemented medium, ability to overcome quiescence induced by serum deprivation, growth at low cell density, and an extended life span in vitro. Furthermore, when explanted in vivo onto the chorioallantoic membrane of chicken embryo, the transformed CEFs expressing v-erbA in addition to v-erbB exhibited a high proliferative rate, giving rise to fibrosarcoma tumors that were ten times larger than those developed from transformed CEFs expressing v-erbB alone. All these data show that CEFs expressing the v-erbA oncogene display activated growth and suggest that the v-erbA product interferes with the mechanisms regulating the growth and/or differentiation of primary CEFs.

1. When injected into irradiated chickens, haemopoietic stem cells give rise to well-defined erythrocytic colonies in the host marrow. Such stem cells (CFU-M = Colony Forming Unit in Marrow) have been found in different tissue of the chicke embryo (yolk sac, blood, marrow). Analysis of the properties of CFU-M reveals that they represent two classes of stem cells: pluripotent stem cells mainly in adult marrow and erythrocytic-committed stem cells present in yolk sac. 2. Yolk sac contains the main pool of CFU-M during the major part of embryonic life. In the blood of 6-day-old embryo, there are three or four times more CFU-Ms than in the yolk sac; they are no longer detected in the blood after the 16th day of incubation. During development of the marrow, stem cells are actively differentiating and their total number remains the same from 16 days to hatching.

We analyzed the expression of the c-erbA proto-oncogene in different tissues of chicken embryos. c-erbA transcripts were found at low levels in the lung, kidney, liver, and heart and in high amounts in embryonic blood cells. Nuclease mapping assays proved that these transcripts were true c-erbA transcripts. In situ hybridization on fractionated embryonic blood cells showed that c-erbA transcripts were predominantly found in erythroblasts, particularly during the final step of differentiation. Life span analysis of c-erbA mRNAs revealed their relative instability, demonstrating that the high level of c-erbA transcripts in embryonic erythroblasts was not the result of passive accumulation. These results suggest that the c-erbA genes play some role in erythrocyte differentiation.

A simultaneous decay of the expression of Im 140 kDa, Im 150 kDa and Im 160 kDa high MW membrane antigens, concomitant with the cell proliferation arrest, was observed during erythropoietin induced differentiation of ts 34 AEV-transformed erythroid cells cultivated at the restrictive temperature. Expression of embryo-immature antigens was maintained during induced differentiation of erythroleukemia cells, but their MW shifted from 50 to 48 kDa, which corresponds to the MW of embryo-immature antigens detected on normal erythroid cells. In the absence of erythropoietin at the restrictive temperature, conditions under which the ts 34 AEV-transformed erythroid cells fail to differentiate and maintain their capacity to proliferate, the expression of high MW antigens as well as the expression of embryo-immature antigens remained unaffected. Therefore, it is shown that the expression of specific membrane antigens is modulated under conditions rendering the erythroleukemia cell differentiation process possible.