The H19 TATA-less promoter is efficiently repressed by wild-type tumor suppressor gene product p53.

The developmentally regulated H19 gene displays several remarkable properties: expression of an apparently non-translated mRNA, genomic imprinting (maternal allele only expressed), relaxation of the imprinting and/or epigenetic lesions demonstrated in some tumors. Despite several observations after relaxation of imprinting status of the gene, data on trans and cis-acting factors required for the human H19 gene expression are still missing. As a first approach to address identification of factors involved in the regulation of the gene, we found that cells from a p53 antisense-transfected HeLa clone displayed increased amounts of H19 transcripts when compared to the non-transfected cells. Moreover, a HeLa clone stably transfected with a temperature sensitive (ts) 143 Ala p53 mutant exhibited temperature-dependent regulation of H19 expression. This preliminary indication of the repressing effect of the p53 protein on H19 expression has been confirmed by transient cotransfection experiments in HeLa cells, using luciferase surrogate constructs under the control of the 823 bp sequence immediately upstream of the transcription start point of the H19 gene, and different constructs containing sense, antisense or a ts 143 Ala mutant p53 cDNA. We observed an increase of H19 promoter-driven activity in transient cotransfections with the antisense p53 cDNA and the temperature sensitive mutant p53 at the non-permissive temperature, but a decrease with sense wild-type p53 cDNA. Furthermore, the cotransfection experiments were repeated in a cell line lacking endogenous p53. (Calu 6 cells) and the results provided additional evidence for a down regulation of the expression of the H19 gene by the p53 protein.

The 5' part of the human H19 RNA contains cis-acting elements hampering its translatability.

H19 is an imprinted gene developmentally regulated in man and mouse and implicated in various neoplasms. No corresponding protein product has yet been detected, although several open reading frames (ORFs) could be identified along its RNA. The largest ORF found in the human gene could encode a putative 26 kDa protein. We have isolated two H19 cDNAs (AP and ES) that contain this ORF4 and correspond to incomplete copies of the unique 2.3 kb H19 RNA. In transient expression assays, AP was able to synthesize a 26 kDa protein whereas ES was not. With respect to ORF4, ES exhibits a 536 bp long GC-rich 5' untranslated region, whereas AP contains the last 22 nucleotides of this 5'UTR. Using deletions and point mutations, we have found that the length and probably the secondary structure of the 5'UTR strongly hampers the translatability of the RNA. In addition, a potential role of upstream ORFs (uORFs) was detected as stressed by the enhances translation of a construct mutated in uORF3 overlapping ORF4. Interactions between H19 and proteins are indicated by a specific binding between 5'UTR derived RNA segments and two nuclear proteins of about 27 kDa. Our results favor a potential role of these particular structures and binding properties in general trans-regulation of RNA post-transcriptional processes rather than in normal control of H19 mRNA translation.

In vivo cooperation of two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, leads to transformation and immortalization of chicken myelomonocytic cells.

To investigate a possible in vivo cooperation between the p61/63myc and P135gag-myb-ets proteins, we used a previously constructed retrovirus, named MHE226, which contains the fused v-myb and v-ets oncogenes of the E26 retrovirus and the v-myc oncogene of MH2. For that purpose, chicken neuroretina cells producing MHE226 and pseudotyped with the Rous associated virus-1 (RAV-1) helper virus were injected in 1-day-old chickens. In control experiments, we also injected chicken neuroretina cells producing E26 (RAV-1), RAV-1 alone, or constructs lacking one of the oncogenes of MHE226. The average life span of MHE226-infected chickens is half that of E26-infected chickens. MHE226-infected chickens harbor tumors scattered in many organs, but compared with E26, MHE226 induced a weak leukemia. Study of integration sites suggests that the majority of the tumors results from clonal or oligoclonal events. Cell cultures were derived from the tumors of MHE226-infected chickens and grown in standard medium without addition of exogenous chicken myelomonocytic growth factor. These cells still divide at high rate after more than 100 passages and can thus be considered immortalized. By using several criteria, these cells were characterized as precursors of the myelomonocytic lineages.

The c-Ha-ras oncogene induces increased expression of beta-galactoside alpha-2, 6-sialyltransferase in rat fibroblast (FR3T3) cells.

Alteration in cell surface carbohydrates, and in particular cell surface sialylation, have been known to occur during oncogenic transformation. To examine the basis for such changes, we have transformed the rat fibroblast cell line FR3T3 with the oncogenes c-Ha-ras EJ, v-mycOK10, v-src, polyoma virus middle T or the transforming bovine papilloma virus 1 (BPV1), and measured the sialytransferase activities of cellular lysates. We found that, in contrast to all other oncogenes examined, c-Ha-ras induced a striking increase in beta-galactoside alpha-2,6-sialytransferase (Gal alpha-2,6-ST) activity in FR3T3 cells. This increase in Gal alpha-2,6-ST activity resulted in the increased expression of cell surface alpha-2,6-linked sialic acid on cell surface glycoconjugates, as determined by cell staining with fluorescein-labelled Sambucus nigra agglutinin. Immunoprecipitation and immunofluorescence experiments revealed that the increase in Gal alpha-2,6-ST activity was due to an elevation of expression of the enzyme. Moreover, Northern analysis suggested that the increased expression of this enzyme was the result of an increase in the steady-state mRNA level of the Gal alpha-2,6-ST gene. These results support the notion that alterations seen in cell surface glycoconjugates during oncogenic transformation can be the result of altered expression of glycosyltransferases.

Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors.

A putative role for the vascular endothelium as target for autoantibodies has been suggested in several autoimmune disorders and connective-tissue diseases. However, there are some difficulties linked to the use of cultured endothelial cells (EC) that limit considerably the extensive studies on the nature of endothelial target antigens involved. To overcome this problem, human EC, derived from umbilical veins, were transfected with recombinant plasmid pSV1 which contained the early genes of simian virus SV40. These transfected cells, called EC-pSV1, are able to grow without EC growth supplement and demonstrate a population doubling time of about 50 h. Among the EC properties, EC-pSV1 retain intracellular content of angiotensin-converting enzyme activity, exhibit constitutive production of interleukin 6 and of a growth-promoting activity on early passage EV, express intercellular adhesion molecule 1 (ICAM-1) and its up-regulation by tumor necrosis factor alpha, but have lost the expression of factor VIII-related antigen. Moreover, EC-pSV1 express a 55-kDa antigen found on EC and human platelets, and presumably acting as an antibody target in some cases of non-allergic asthma. However, at the 50-55th generation, morphological changes and altered growth behavior were visible. This work demonstrates that transfection of EC with SV40 T antigens may be of interest, particularly in areas of research including the study of EC targets involved in different human diseases.

Comparison of sialyl- and alpha-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased beta-galactoside alpha-2,6-sialyltransferase.

Previous studies have indicated that transfection of NIH3T3 cells with the ras oncogene induced modifications of the terminal glycosylation of N-linked glycans which appeared in the early stage after transfection. These changes affected especially the terminal part of N-linked glycans which is substituted with alpha-1,3-Gal residues in NIH3T3 and with Neu5Ac residues in the ras-transformed counterpart. We have transformed NIH3T3 cells with the human c-Ha-ras oncogene, evaluated tumorigenicity and metastatic capacity in vivo and compared alpha-1,3-galactosyltransferase, alpha-2,3- and alpha-2,6-sialyltransferases activities. By using different specific acceptors, we detected the enhancement of sialic acid transfer in transformed cells while the activity of alpha-1,3-galactosyltransferase remained unchanged. We showed that the higher sialyltransferase activity was due to the increase of beta-galactoside alpha-2,6-sialyltransferase in ras-transfectant although alpha-2,3-sialyltransferase was weakly expressed in these cells. On the basis of binding of different lectins, we correlated these observations with changes of protein glycosylation. We concluded that altered glycosylation of ras-transformed NIH3T3 is the result of a competitive effect of the enzymes acting for terminal glycosylation of N-linked glycans and the reflection of the higher expression of alpha-2,6-sialyltransferase.

[Modifications of sialylation in BHK 21/C13 cells after in vitro transfection by c-Ha-ras human oncogene]. / Modifications de la sialylation des cellules BHK 21/C13 après transfection in vitro par l'oncogène humain c-Ha-ras.

A Hamster kidney fibroblast cell line (BHK 21/C13) has been transfected by c-Ha-ras human oncogene. The expression of the oncogene significantly modified the global sialytransferase activity of the cell extract. This activity is enhanced on an average 2.5 fold whatever the acceptor. In addition, the proportion of cell-surface associated N-glycolylneuraminic acid is enhanced in transfectants, the ratio N-acetylneuraminic acid/N-glycolylneuraminic acid decreases from 7.10 to 2.80. These results suggest that a tight relationship exists, in c-Ha-ras transfected BHK cells, between the expression of the oncogene and the neuraminic acid metabolism as well as a gene regulation of glycoconjugate sialylation.

Rapid and transient expression of Ets2 in mature macrophages following stimulation with cMGF, LPS, and PKC activators.

We reported previously that Ets2 is expressed in normal and transformed macrophages. We show here that the expression of both c-ets-2 mRNA proteins is induced rapidly and transiently in chicken nondividing bone marrow-derived macrophages but not in E26-transformed myeloblasts in response to chicken myelomonocytic growth factor (cMGF), an avian hematopoietic growth factor required for survival, proliferation, and colony formation of avian myeloid cells. c-ets-2 expression is also rapidly induced in chicken bone marrow-derived macrophages, human monocytes, and mouse peritoneal macrophages in response to LPS and/or PKC activators. The rapid induction of Ets2 after treatment of chicken bone marrow-derived macrophages by cMGF is blunted after down-regulation or inactivation of PKC, suggesting a role of PKC in the cMGF-induced signal transduction pathway. Because Ets2 is localized in the nucleus of macrophages and binds to DNA in vitro, the kinetics of its expression suggest a role for Ets2 in the transduction within the nucleus of specific signals received at the cell membrane and involved in securing the survival and/or the development of functional competence of these cells.

A single amino-acid substitution in the DNA-binding domain of the myb oncogene confers a thermolabile phenotype to E26-transformed myeloid cells.

A biologically active provirus of the ts 143 E26 mutant that is temperature-sensitive (ts) for myeloblast transformation was molecularly cloned. The predicted amino-acid sequence of the v-myb-encoded domain of the mutant P135gag-myb-ets protein displayed two single amino-acid changes, one of which was non-conservative when compared to the wild-type E26 v-myb sequence. This mutation, which substitutes a threonine residue (wild-type) for an arginine residue (mutant), is located within the amino-terminal part of v-myb in the DNA-binding domain at a position which is conserved between the c-myb genes of chicken, humans, mice and Drosophila. Introduction of this mutation into the genome of a wild-type E26 virus was sufficient to induce a ts phenotype similar to that obtained with the original ts 143 E26 virus.

The human c-myc exon 1 product: preparation of antisera and analysis of its expression.

We investigated the coding capacity of the previously reported open reading frame (ORF) of the human c-myc exon 1. By in vitro translation assay, we found that exon 1 ORF was translated into a 20-kd protein (p20 protein). In order to obtain antisera raised against the p20 protein, we constructed a plasmid vector which expressed most of the exon 1 ORF as a 25-kd (P25) protein in Escherichia coli. Polyclonal antisera raised against this P25 protein specifically precipitated a chimeric protein which contained exon 1-related amino acid sequences. We used these antisera to test for the existence of an exon 1 product in human cells. In the human cell lines tested, these antisera have failed so far to detect any exon 1-related proteins. However, exon 1-related proteins were detected with the anti-p20 antisera in quail embryonic cells (QEC) transfected by human c-myc recombinants constructed to express such proteins, but were expressed at low levels compared with the human c-myc protein also expressed in the transfected QEC. Our results suggest that secondary structure of the mRNA could be responsible for the low expression of the exon 1 product in QEC.

Identification in chicken macrophages of a set of proteins related to, but distinct from, the chicken cellular c-ets-encoded protein p54c-ets.

Using an antiserum to a bacterially expressed polypeptide corresponding to 56 amino acids of v-ets, we previously identified in chicken tissues a protein of 54 kd (p54c-ets) which shares extensive sequence homology to the v-ets-encoded domain of the E26-transforming protein p135gag-myb-ets and is thus apparently encoded by the c-ets proto-oncogene. We report here that the anti-ets serum specifically identifies in chicken cells a second set of proteins of 60 kd (p60), 62 kd (p62) and 64 kd (p64) which appear to be highly related to each other but display only a limited domain of homology with p54c-ets and p135gag-myb-ets and are thus probably encoded by a gene(s) partially related to, but different from c-ets. In contrast to p54c-ets which is expressed at high levels in chicken lymphoid tissues, prominent syntheses of p62 and p64 were found in both normal and transformed chicken macrophages but not in avian cells corresponding to immature stages of the myeloid differentiation pathway. These observations together with the fact that differentiation of avian myeloblastosis virus-transformed myeloblasts into macrophage-like cells after treatment with 12-O-tetradecanoylphorbol-13-acetate is accompanied by the synthesis of p62 and p64 suggest a role for these proteins in chicken macrophage differentiation or function. Induction of differentiation of human leukemia cell lines HL60 and U937 into macrophages is also accompanied by the increased synthesis of c-ets-encoded 68 kd, 62 kd and 58 kd proteins.

Transformation of quail embryo fibroblasts by a retrovirus carrying a normal human c-myc gene.

We have constructed avian retroviruses expressing the human c-myc oncogene. These viruses morphologically transformed primary quail embryo fibroblasts upon transfection and infection. Transformed cells produced viruses harboring a spliced c-myc gene and contained high levels of p64-67c-myc protein. One of these infectious viruses, vSX-AHM, was molecularly cloned and the nucleotide sequence of the spliced c-myc insert determined. No mutation was found within the c-myc coding sequence of this transforming clone when compared to the normal genomic progenitor. Thus, we concluded that no mutation within the human c-myc gene is required to induce primary avian embryo fibroblast transformation.

Increased transcription of the c-myc oncogene in two methylcholanthrene-induced quail fibroblastic cell lines.

The expression of three c-onc genes (c-erb, c-myc, c-myb) was investigated in five cell lines established from fibrosarcomas induced with 20-methylcholanthrene (MCA) of Japanese quails. These cell lines showed low levels of the three c-onc genes, with the exception of two cell lines that accumulated moderate (MCAQ 1-4) and large amounts (MCAQ3-5) of c-myc RNA. Molecular cloning and restriction endonuclease analyses indicated that expression of c-myc in these two cell lines were not associated with detectable rearrangements in the c-myc locus, that the size of the c-myc transcript (2.7 kb) in MCAQ 3-5 was similar to that of the normal c-myc messenger RNAs (mRNA) and that the transcriptional activation observed in MCAQ 3-5 was not mediated by the LTR (long terminal repeat) of a proximate ALV (avian leukosis virus) provirus. Finally, when analysed with the restriction enzymes Msp I and Hpa II, the c-myc locus of MCAQ 3-5 and MCAQ 1-4 was found hypomethylated as compared with that of the other cell lines tested that show low levels of c-myc transcripts. Our results suggest that one of the ways methylcholantrene could mediate transformation is by inducing an abnormal regulation of the c-myc gene.

The cellular oncogenes c-myc, c-myb and c-erb are transcribed in defined types of avian hematopoietic cells.

The possible role of normal chicken cellular sequences c-erb, c-myb and c-myc, together referred to as c-onc genes and related to the oncogenes of defective avian acute leukemia retroviruses (DLVs), was investigated by determining the accumulation of c-onc RNA in different avian cells an cell lines. Levels of c-myc and in some instances c-myb RNA are elevated in immature hematopoietic cells or cell lines from various lineages but more mature hematopoietic cells, as well as non-hematopoietic cells, contain only low levels. In contrast, the level of c-erb RNA is generally low, but high in a small number of normal bone marrow cells. The results indicate that the cellular homologues of the viral oncogenes are differentially expressed during hematopoiesis. They also indicate that the hypothesis that DLV target cells express their homologous c-onc genes might hold for c-erb, but is not valid in its simple form for c-myc and c-myb.

A putative second cell-derived oncogene of the avian leukaemia retrovirus E26.

The acute avian leukaemia retroviruses AMV and E26 both induce myeloblastosis in vivo and transform myeloblasts in vitro. Both viruses contain the oncogene v-myb first described for AMV. Unlike AMV, E26 has the additional capacity to induce erythroblastosis in vivo and to transform erythroblasts. Previous analyses indicated that the genome of E26 also contained nucleotide sequences distinct from v-myb and unrelated to viral replicative genes. Using a molecularly cloned E26 provirus, we have now identified a novel nucleotide sequence designated v-ets (for E-twenty-six specific) of approximately 1.5 kilobase pairs (kbp) located next to v-myb. v-ets possesses all the structural characteristics of a putative new oncogene: it has a conserved cellular counterpart c-ets which is transcribed in some normal chicken cells as a major 7.5-kb polyadenylated RNA. Although our results now await elucidation of their biological significance, we propose that v-ets could be a new oncogene accounting for the additional transforming properties of E26, or potentiating the transforming properties of the v-myb oncogene.

Two different types of transcription for the myelocytomatosis viruses MH2 and CMII.

The four avian defective leukemia retroviruses (DLVs) MC29, CMII, MH2 and OK10 all transform primarily macrophages in an in vitro bone marrow transformation assay, and contain specific nucleotide sequences closely related to the myc gene of MC29. These viruses were thought to express their oncogenic potential through a gag-myc fusion polyprotein, since fusion polyproteins were found in all tested cells transformed by MC29. We show here that MH2 virus does not conform to this model. Whereas MC29 produces only one mRNA detectable by RNA blotting in productively transformed cells, we reported recently that OK10 induced the synthesis of two myc-containing mRNAs, the smaller species being a spliced mRNA and a possible candidate for a transforming protein lacking gag determinants. However, the studies with OK10 were ambiguous because this virus produced also, in infected cells, a fusion protein containing gag, pol and myc determinants. We have therefore investigated the transcription pattern of the two other members of this group of viruses, namely CMII and MH2. Our results show that CMII resembles MC29 whereas MH2 produces, as OK10, two mRNAs containing myc-related sequences. However, unlike OK10, the MH2 fusion protein of 100 kd described previously cannot contain myc determinants and thus is likely to produce from its subgenomic mRNA a v-myc protein-lacking gag determinants. We thus conclude that the product of the v-myc oncogene is transforming with (MC29) or without (MH2) its fusion to gag determinants and that the multiple oncogenic spectrum is not basically affected since MH2 and MC29 both transform macrophages, fibroblasts and epithelial cells.

Characterization of the oncogene (erb) of avian erythroblastosis virus and its cellular progenitor.

Avian erythroblastosis virus (AEV) induces primarily erythroblastosis when injected intravenously into susceptible chickens. In vitro, the hematopoietic target cells for transformation are the erythroblasts. Occasional sarcomas are also induced by intramuscular injection, and chicken or quail fibroblasts can be transformed in vitro. The transforming capacity of AEV was shown to be associated with the presence of a unique nucleotide sequence denoted erb in its genomic RNA. Using a simplified procedure, we prepared radioactive complementary DNA (cDNAaev) representative of the erb sequence at a high yield. Using a cDNAaev excess liquid hybridization technique adapted to defective retroviruses, we determined the complexity of the erb sequence to be 3,700 +/- 370 nucleotides. AEV-transformed erythroblasts, as well as fibroblasts, contained two polyadenylated viral mRNA species of 30 and 23S in similar high abundance (50 to 500 copies per cell). Both species were efficiently packaged into the virions. AEV-transformed erythroblasts contained additional high-molecular-weight mRNA species hybridizing with cDNAaev and cDNA5' but not with cDNA made to the helper leukosis virus used (cDNArep). The nature and the role, if any, of these bands remain unclear. The erb sequence had its counterpart in normal cellular DNA of all higher vertebrate species tested, including humans and fish (1 to 2 copies per haploid genome in the nonrepetitive fraction of the DNA). These cellular sequences (c-erb) were transcribed at low levels (1 to 2 RNA copies per cell) in chicken and quail fibroblasts, in which the two alleged domains of AEV-specific sequences corresponding to the 75,000- and 40,000-molecular-weight proteins seemed to be conserved phylogenetically and transcribed at similar low rates.

Three new types of viral oncogene of cellular origin specific for haematopoietic cell transformation.

The RNAs of seven replication-defective leukaemia virus (DLV) strains contain three types of unique sequences, which correlate with the capacity of a given virus strain to transform erythroblasts, macrophage-like cells and myeloblasts, respectively. These sequences, termed erb, mac and myb, have their counterparts in the normal DNA of avian and mammalian species. Our results indicate that DLVs represent recombinants between a common 'vector' related to a chicken endogenous virus and one of three types of cellular gene possibly involved in haematopoietic differentiation.