Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus

Chicken erythroblast cell strains and a cell line transformed by ts mutants of avian erythroblastosis virus (AEV) terminally differentiate when shifted to the nonpermissive temperature (42°C). The differentiated cells resemble mature erythrocytes with respect to morphology and ultrastructure, expression of differentiation-specific cell-surface antigens, pattern of protein synthesis and hemoglobin content. Terminal differentiation is dependent on conditions favoring the differentiation of normal erythroid progenitor cells, including an erythropoietin-like factor. Colonies of ts AEV cells grown at 42°C in semisolid medium resemble erythrocyte colonies derived from normal erythroid progenitor cells. The colonies obtained were comparable in size or slightly larger than the late erythroid precursor (CFU-E) colonies. These results suggest that AEV-transformed cells are blocked at a stage of differentiation that is more advanced than that of the uninfected target cells. ts AEV cells are irreversibly committed to terminal differentiation within 20 to 30 hr after shift to 42°C.     

Mitogenic action of factors secreted by avian erythroblastosis virus transformed cells

We describe here the capacity of erythroid LSCC HD3 cells, transformed with a ts mutant of avian erythroblastosis virus, to grow in a chemically defined medium without serum at 36 degrees C, but not at 41 degrees C. At this latter temperature the activity of v-erbB oncogene is suppressed. However, cell growth at 41 degrees C could take place either by addition of the medium derived from LSCC HD3 cells grown at 36 degrees C (conditioned medium), or by addition of fetal calf serum. These results show that LSCC HD3 cells, maintained under conditions in which the v-erbB oncogene is active, secrete growth factor(s) which exhibit a mitogenic effect similar to that observed with calf serum.     

Target cells infected by avian erythroblastosis virus differentiate and become transformed

Transformation in vitro of bone marrow cells by avian erythroblastosis virus (AEV) gives rise to rapidly growing cells of erythroid nature. Target cells of neoplastic transformation by AEV are recruited among the early progenitors of the erythroid lineage, the burst-forming units-erythroid (BFU-E). They express a brain-related antigen at a high level and an immature antigen at a low level. We show that AEV-transformed cells express low levels of the brain antigen and high levels of the immature antigen. Their response to specific factors regulating the erythroid differentiation indicates that they are very sensitive to erythropoietin. Furthermore, cells transformed by a temperature-sensitive mutant of AEV differentiate into hemoglobin-synthesizing cells 4 days after being shifted to the nonpermissive temperature. All these properties are similar to those of late progenitors of the erythroid lineage, the colony-forming units-erythroid (CFU-E). These results indicate that the AEV-transformed cells are blocked in their differentiation at the CFU-E stage.     

Expression of differentiation and age-related antigens on chicken erythroleukemia cells transformed by avian erythroblastosis virus (AEV)

Immature circulating chicken red cells express on their surface two antigenic molecules referred to as Im 48 kD and Im 140 kD antigens. The Im 140 kD antigen is not present beyond the erythroblast stage while the expression of Im 48 kD antigenic molecule remains detectable on circulating erythrocytes of embryos and young chickens, but not on erythrocytes of adult animals. In addition to Im 48 kD and Im 140 kD antigens, the avian erythroblastosis virus (AEV)-transformed erythroid cells express two novel high molecular weight (MW) immature antigens referred to as Im 150 kD and Im 160 kD. Since the transformed erythroid cells are apparently blocked at a stage close to the colony-forming units erythrocytic (CFU-E), these molecules might be expressed on these progenitor cells. The age-related antigenic molecules referred to as E1 48 kD and A 40 kD/A 85 kD antigens are detected on erythrocytes of embryos (and young chickens) and adult animals respectively. The E1 48 kD antigen as well as an antigen related to the A 40 kD were also detected on AEV-transformed erythroid cells deriving from both young chicken bone marrow and yolk sac. The presence of an adult antigen on the embryonic cells might well be related to the transformation by AEV, since the yolk sac CFU-E progenitor cells do not bear the adult antigenicity.     

The methylation state of the proviruses in avian sarcoma virus transformed chick and rat cells

The endogenous viruses in the avian cells are not completely methylated, nor are the Schmidt-Ruppin RSV-D (SRD) proviruses in the infected cells completely unmethylated. Avian sarcoma proviruses integrated in rat transformed cloned cells are heavily methylated. In these cells, a region in the 3' end of the env gene is unmethylated in all the src-containing proviruses but not in the transformed defective (td) proviruses. A possible role for the hypomethylation of the 3' end of the env region is proposed.     

Molecular cloning and characterization of the chicken DNA locus related to the oncogene erbB of avian erythroblastosis virus.

Chicken cell DNA contains sequences which are homologous to the avian erythroblastosis virus oncogene v-erb. These cellular sequences (c-erb) have been isolated from a library of chicken cell DNA fragments generated by partial digestion with AluI and HaeIII and shown to be shared by at least two loci in the chicken DNA. One of them, denoted c-erbB, contains approximately 1.8 kilobase pairs of chicken DNA homologous to the 3' part of the v-erb oncogene (v-erbB). Restriction mapping studies show that the c-erbB DNA sequences homologous to v-erbB are distributed among six EcoRI fragments located in a single genomic region. Heteroduplexes between v-erbB in viral RNA and cloned c-erbB DNA show that the chicken DNA sequences homologous to v-erbB are interrupted by 11 DNA sequences not present in the v-erb oncogene. We conclude from our data that the c-erbB locus might represent the cellular progenitor for the v-erbB domain of the v-erb oncogene.     

Isolation and characterization of chicken DNA homologous to the two putative oncogenes of avian erythroblastosis virus

The genome of avian erythroblastosis virus contains two independently expressed genetic loci (v-erbA and v-erbB) whose activities are probably responsible for oncogenesis by the virus. Both loci are closely related to nucleotide sequences found in the DNA and RNA of chickens and other vertebrates. We have isolated and characterized chicken DNA homologous to v-erbA and v-erbB. The two viral genes are represented by separate domains within chicken DNA (c-erbA and c-erbB), which are separated by a minimum of 12 kilobases (kb) of DNA and may not be linked at all. The nucleotide sequences shared by the viral and cellular erb loci are colinear, but the cellular loci are interrupted by multiple intervening sequences of various lengths. Polyribosomes prepared from normal chicken embryos contain two polyadenylated RNAs transcribed from c-erbA and two transcribed from c-erbB. The evident coding regions of these RNAs represent an unusually small fraction of the lengths of the RNAs, as if the 3′ untranslated domains of the RNAs might be exceptionally large (3–11 kb). These findings indicate that the c-erb loci are normal vertebrate genes rather than genes of cryptic endogenous retroviruses, and that they may have a role in the metabolism of normal cells. It appears that the viral erb genes, like most other retrovirus oncogenes, have been copied from cellular genes. In the viral genome, the two genes are devoid of introns, but they remain independently expressed loci, and they remain colinear with the coding domains of their cellular progenitors.     

Cell-free translation of avian erythroblastosis virus RNA

Avian erythroblastosis virus (AEV) RNA rescued from nonproducer cells by superinfection with a helper virus is translated into three polypeptides in the messenger-dependent rabbit reticulocyte lysate. A 75,000 molecular weight polypeptide (P75AEV) is synthesized from 28S RNA and is encoded by the 5' section of the AEV RNA, including gag-related and AEV-specific sequences. The P75AEV synthesized in infected cells and the P75AEV synthesized in the cell-free system are electrophoretically identical. A 44,000 molecular weight polypeptide (P44AEV) is synthesized from 20-24S RNA, apparently from the 3' section of the AEV-specific RNA sequence. A minor 37,000 molecular weight polypeptide (P37AEV) is synthesized from 20S AEV RNA. A comparison is drawn between the cell-free products of MC29 and AEV RNAs.     

Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

Isolation and characterization of multiple human genes homologous to the oncogenes of avian erythroblastosis virus

Human DNA sequences complementary to the oncogenes v-erbA and v-erbB of avian erythroblastosis virus have been isolated from a genomic DNA library. Two clones, lambda he-A1 and lambda he-A2, were related to the erbA gene and one to the erbB gene (lambda he-B). The two erbA genes were only distantly related to each other as judged from hybridization analysis. Furthermore, human chromosomal DNA appears to contain one or two additional genes analogous to the lambda he-A2 sequence, whereas the mouse genome contained only two genes complementary to lambda he-A1 and lambda he-A2, respectively. Polyadenylated RNA species, 5.0 kb in size, were found in the human HeLa and the human hematopoietic K562 cell lines, suggesting that at least some of the erb-related genes are active and do not represent pseudogenes. Taken together, the data demonstrate that two distantly related classes of erbA genes exist in human and mouse DNA, and that multiple copies of genes belonging to one of these two classes exist in the human genome.     

DNA methylation: organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes.

The restriction enzymes HhaI and HpaII, whose activity is inhibited by cytosine methylation within their recognition sites, have been utilised as probes to study methylation in the vicinity of the ovalbumin gene in DNA from various chicken tissues. This was complemented by a preliminary study of methylation in the regions of chicken ovotransferrin (conalbumin), ovomucoid and beta-globin genes. From our data we conclude that HaI or HpaII sites can be divided in 3 classes according to their pattern of methylation in different tissues. In the first class of sites (mV class) the extent of methylation varies in different tissues. The patterns obtained show that methylation at the sites located within and around the 3 genes which code for egg white proteins is in general lowest in oviduct of laying hen, where these genes are expressed. However some sites are not methylated (m- class) and others are 95 to 100% resistant (m+ class) to digestion by HhaI or HpaII in the DNAs of all the tissues which were tested. Our study has also revealed a remarkable number of allelic variants for the presence of HhaI or HpaII sites in the region of the ovalbumin gene.     

Expression of endogenous avian myeloblastosis virus information in different chicken cells.

Uninfected chicken cells were found to contain endogenous avian myeloblastosis virus (AMV)-specific information. Different tissues from chicken embryos and chickens expressed different amounts of the AMV-specific information. The endogenous AMV-related RNA was most abundant in bone marrow cells, which contained about 20 copies per cell. About 5 to 10 copies of AMV endogenous RNA per cell were found in embryonic yolk sac cells and bursa cells. The spleen, muscle, liver, and kidney cells of chickens and the fibroblasts of chicken embryos contained about two copies per cell. The amounts of AMV endogenous RNA in bone marrow, yolk sac, and bursa varied with age. From 19-day-old embryos to 2-week-old chickens, the bone marrow contained 20 copies of AMV RNA per cell. Bone marrow cells from 2-year-old chickens contained five copies per cell. Yolk sac cells of 10-day-old embryos and 1-day-old chickens were found to contain two copies per cell, whereas in 15- to 17-day-old embryos, these cells contained 5 to 10 copies. These results indicate that the level of endogenous AMV expression correlates with the development of granulopoiesis of the chicken hemopoietic system. The results of experiments on the thermostability of RNA-DNA hybrids indicated that the endogenous AMV RNA is closely related to viral AMV RNA. The expression of endogenous AMV information is independent of the activity of the chick helper factor. This endogenous AMV information is expressed as 20 to 21S RNA in both bone marrow and yolk sac cells.     

Dissecting the activating mutations in v-erbB of avian erythroblastosis virus strain R.

The v-erbB oncogene isolated from the R (or ES4) strain of avian erythroblastosis virus is capable of inducing erythroleukemia and fibrosarcomas. This oncogene differs from the proto-oncogene c-erbB, the avian homolog of the epidermal growth factor receptor, by its lack of an intact ligand-binding domain as well as additional alterations in its cytoplasmic coding sequences. By contrast, the insertionally activated c-erbB, a variant oncogene, which encodes a product that also lacks the ligand-binding domain but is otherwise unaltered in its cytoplasmic coding sequences, is capable of inducing leukemia but cannot induce sarcomas. In this report, we show that the critical changes for activating the sarcomagenic potential displayed by v-erbB R are two point mutations within the tyrosine kinase domain and an internal deletion of 21 amino acids in the carboxyl-terminal regulatory domain. The removal of the carboxyl-terminal autophosphorylation sites is not obligatory. These activating mutations (Arg-263 to His, Ile-384 to Ser, and the deletion of residues 494 to 514), when introduced singly into the insertionally activated c-erbB, all dramatically increase fibroblast-transforming potential. Arg-263 resides near the highly conserved HRD motif of the kinase domain, and its mutation to His increases the autophosphorylation activity. The other two mutations do not alter the intrinsic kinase activity and presumably affect other aspects of the receptor involved in growth signaling. Therefore, the high transforming potential of v-erbB R is a consequence of synergism among multiple activating mutations.     

Analysis of integrated avian RNA tumor virus DNA in transformed chicken, duck and quail fibroblasts.

The state of integration of avian sarcoma virus DNA in the genomes of transformed chicken, duck, and quail fibroblasts was deduced by means of restriction enzyme digestion of total cell DNA, gel electrophoresis, and subsequent analysis by the procedure of Southern. The cells used in these studies were either mass-infected cultures or clones of infected cells selected by their ability to form colonies in agar. For both mass-infected cultures and clones of cells of all three species, we found that integration occurred at a specific site on the viral genome but appeared to occur at many sites on the cell genome. At least some of the integrated viral DNA existed as intact nonpermuted species flanked by direct terminal repeats of at least 0.134 megadalton (217 base pairs). For each of 12 transformed quail clones studied, it was possible to detect, after digestion with Kpn I, unique junctions between viral and cellular DNA. That is, at our level of analysis, the integration site on the cell genome for each clone was different. However, within each of the 17 chicken and 9 duck clones of transformed cells, a heterogeneity presumably occurred during the outgrowth of the cell clone population, in that we could not readily detect identifiable cell-virus junction fragments.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.