Evidence for multiple sequences and factors involved in c-myc RNA stability during amphibian oogenesis.

To investigate the molecular mechanisms regulating c-myc RNA stability during late amphibian oogenesis, a heterologous system was used in which synthetic Xenopus laevis c-myc transcripts, progressively deleted from their 3' end, were injected into the cytoplasm of two different host axolotl (Ambystoma mexicanum) cells: stage VI oocytes and progesterone-matured oocytes (unfertilized eggs; UFE). This in vivo strategy allowed the behavior of the exogenous c-myc transcripts to be followed and different regions involved in the stability of each intermediate deleted molecule to be identified. Interestingly, these specific regions differ in the two cellular contexts. In oocytes, two stabilizing regions are located in the 3' untranslated region (UTR) and two in the coding sequence (exons II and III) of the RNA. In UFE, the stabilizing regions correspond to the first part of the 3' UTR and to the first part of exon II. However, in UFE, the majority of synthetic transcripts are degraded. This degradation is a consequence of nuclear factors delivered after germinal vesicle breakdown and specifically acting on targeted regions of the RNA. To test the direct implication of these nuclear factors in c-myc RNA degradation, an in vitro system was set up using axolotl germinal vesicle extracts that mimic the in vivo results and confirm the existence of specific destabilizing factors. In vitro analysis revealed that two populations of nuclear molecules are implicated: one of 4.4-5S (50-65 kDa) and the second of 5.4-6S (90-110 kDa). These degrading nuclear factors act preferentially on the coding region of the c-myc RNA and appear to be conserved between axolotl and Xenopus. Thus, this experimental approach has allowed the identification of specific stabilizing sequences in c-myc RNA and the temporal identification of the different factors (cytoplasmic and/or nuclear) involved in post-transcriptional regulation of this RNA during oogenesis.

Evidence for introduction of a variable G1 phase at the midblastula transition during early development in axolotl.

After fertilization in axolotl, the synchronous cell cleavages are triphasic (S, G2 and M phases). Midblastula transition (MBT) begins at the ninth cleavage and is the consequence of lengthening of cell cycles. By spectrofluorometry and incorporation of 3H thymidine into the nuclear DNA followed by autoradiography on individual cells, the time at which a G1 phase appears during early development was investigated. The present results show that the G1 phase was introduced for the first time at MBT and its duration was variable from one blastomere to another. This variability could account for lengthening of cell cycles and be required for zygotic transcriptions necessary for DNA replication. From this point of view, axolotl represents an interesting alternative amphibian model to identify regulators involved in the G1-S transition at MBT during early development.

Post-transcriptional control of c-myc RNA during early development analyzed in vivo with a Xenopus-axolotl heterologous system.

We have set up a heterologous in vivo system to study gene regulation at the post-transcriptional level during early development. This system uses two amphibian species, Xenopus laevis and Ambystoma mexicanum (axolotl), the development of which is three to four times slower than that of X. laevis. The stability of three different synthetic X. laevis c-myc transcripts was followed after injection into fertilized axolotl eggs. One transcript is 2.2 kilobases (kb) long (full-length). The second is 1.5-kb long with most of the 3' untranslated region (3'UTR) removed, and the third corresponds to the 3'UTR (0.7-kb). The behavior of the endogenous axolotl c-myc RNA was compared with the exogenous injected c-myc transcripts. Our results show the existence of several developmental timers controlling degradation of the c-myc molecules. The first is activated at oocyte maturation and affects both the endogenous and exogenous (2.2- and 1.5-kb) transcripts containing the coding regions. A second timer could be linked to the number of cell divisions since fertilization (6th-7th cleavages) and involves the endogenous c-myc RNAs. Another timer could involve the c-myc mRNA molecule itself, because when injected into axolotl eggs, the half-life of the 2.2-kb X. laevis transcript appears to be independent of the axolotl context. After injection into axolotl fertilized eggs, the behavior of this X. laevis full-length c-myc molecule reveals an unexpected increase in the intensity of its autoradiographic signals. This increase occurs independently of events linked to mid-blastula transition and preliminary investigations are discussed.

Differential stability of Xenopus c-myc RNA during oogenesis in axolotl Involvement of the 3' untranslated region in vivo.

We have used the axolotl oocyte (Ambystoma mexicanum Shaw) to study the stability of exogenously injected Xenopus RNAs. Three different cellular developmental stages have been analysed: (1) the growing oocyte (stage III-IV of vitellogenesis), (2) the full-grown oocyte at the end of vitellogenesis (stage VI) and (3) the progesterone-matured stage VI oocyte. Three exogenous RNAs have been synthesized in vitro from a c-myc Xenopus cDNA clone. One transcript is 2.3 kb long (full length), the second is 1.5 kb long, with most of the 3' untranslated region (3'UTR) removed, and the third corresponds to the 3'UTR (0.8 kb). After injection or coinjection of these exogenous Xenopus RNAs into axolotl oocytes, the stability of the molecules was studied after 5 min, 6 h and 21 h by extraction of total RNA and Northern blot analysis.Results show a difference in Xenopus RNA stability during axolotl oogenesis. In growing oocytes, the three synthetic transcripts are gradually degraded. The absence of the 3'UTR is not therefore sufficient to stabilize the transcript during early oogenesis. No degradation is observed in full-grown oocytes, suggesting the existence of stabilizing factors at the end of oogenesis. When stage VI oocytes are induced to mature by progesterone, only the 2.3 and 1.5 kb Xenopus RNAs disappear. This suggests a role for germinal vesicle breakdown in this degradation process as well as the existence of a factor present in the nucleus and involved in the specific destabilization of these RNAs after oocyte maturation. This degradation might implicate several destabilizing sequences localized in the coding or in the 3'UTR of the c-myc gene. In contrast, the 0.8 kb transcript (3'UTR) is not degraded during this period and remains very stable. Therefore, degradation appears distinct from one transcript to another and from one region to another within the same molecule. During maturation, the behaviour of the 2.3 and 1.5 kb transcripts is different when coinjected with the 3'UTR, suggesting a role in trans of this untranslated molecule in c-myc stability. Our approach allows us to analyse the role of the coding and 3'UTR regions of the c-myc RNA in the control of mRNA degradation in vivo.

Evidence for a nuclear factor involved in c-myc RNA degradation during axolotl oocyte maturation.

We have previously described an in vivo heterologous system which has allowed us to study the stability of different Xenopus c-myc RNA constructs injected into axolotl oocytes. In full-grown oocytes, degradation of c-myc RNA does not occur. In mature oocytes treated with progesterone, transcripts containing the coding sequence of the gene are degraded, whereas those corresponding to the 3'UTR (untranslated region) alone are stable. In order to study the role of nuclear or cytoplasmic components in this process, degradation of injected c-myc transcripts was analysed (i) after inhibition of germinal vesicle breakdown (GVBD) in progesterone treated oocytes (ii) after induced maturation of enucleated oocytes, (iii) injection of nuclear contents into immature oocytes and (iv) after direct injection into the germinal vesicle of full-grown oocytes. Our results demonstrate the role of a nuclear factor stockpiled in the germinal vesicle of full-grown oocytes and specifically involved in the degradation of c-myc transcripts containing the coding region. Further biochemical characterization of this new nuclear component should lead to a better understanding of the post-transcriptional control of c-myc expression during oogenesis and early development.

Localization of ras proto-oncogene expression during development in Xenopus laevis.

The expression of the ras protooncogene was investigated in Xenopus laevis, throughout development, by in situ hybridization using a 35S-labelled antisense RNA probe. During oogenesis, the ras RNA was strongly expressed in the cytoplasm of previtellogenic oocytes and further diluted between yolk platelets; no specific localization of transcripts was observed. The signal density was particularly weak over embryo sections until the tailbud stage. On the other hand, a high level of ras RNA expression was detected on sections through the young tadpoles. An intense labelling was observed in several areas, including the branchial apparatus, gut, somites, nervous system, and lens. It is noteworthy that the heterogeneity of labelling increases as tadpoles grow older. Together, these results are discussed in relation to cellular events appearing throughout the early development.

Characterization and expression of a Xenopus ras during oogenesis and development.

We have characterized a cDNA which contains the entire coding sequence of a Xenopus laevis ras protein. The deduced amino acid sequence reveals a strong homology (92%) to human Ki-ras 2B protein. ras expression has been studied both qualitatively and quantitatively during Xenopus development. ras is expressed as a maternal mRNA in oocytes and early embryos at a level up to 1.5 x 10(7) copies per mature oocyte, corresponding to the level of ras mRNA found in 4 x 10(5) somatic growing cells. This level remains constant throughout the first rapid cleavage stages of the blastula before the midblastula transition (MBT). After this stage, the amount of ras RNA decreases gradually until the hatching tadpole stage, when a new zygotic expression is detected in the embryo. From that stage, a constitutive amount of 30-50 ras RNA transcripts per embryonic cell is registered, as observed in Xenopus proliferative somatic cells. The 23-kDa Xenopus ras protein has also been identified by both specific monoclonal antibody and in vitro transcription-translation experiments. It is expressed in oocytes before maturation, indicating that maturation is not the trigger for ras expression. The expression of Xenopus ras at a high level during oogenesis and early development suggests a major function of this gene both in meiosis and in mitosis events during embryonic development.

Genes and mechanisms involved in early embryonic development in Xenopus laevis.

Our laboratory is studying genes involved in the regulation of the balance between cell growth and differentiation during embryonic development in Xenopus. We have analyzed the developmental expression of the proto-oncogenes c-myc, and KiRas 2B, the proliferating cell nuclear antigen (PCNA), and the tumor suppressor gene p53. These genes, usually expressed during cell proliferation, are expressed in the oocyte in large quantities, but the majority of their maternal RNAs are degraded by the gastrula stage. The expression of c-myc and the localization of the protein indicate that c-myc has the characteristics expected for a gene involved in the regulation of the mid-blastula transition, when zygotic expression is turned on in the embryo. Its expression during late development or during regeneration indicates that it enables the cells to remain competent for cycling during organogenesis. In vitro systems that reproduce the principal cellular functions during early development are used as model systems to understand the mechanisms involved in early embryogenesis.

Proto-oncogenes and embryonic development.

The role of proto-oncogenes in embryonic development was investigated using one of the most characterized vertebrates, the amphibian Xenopus laevis. Genes which belong to the major proto-oncogene families have been detected in Xenopus genome. The developmental control of the myc gene was assayed using a characterized Xenopus myc probe and specific antibodies. The myc gene is highly expressed as a stable maternal mRNA in oocyte, and an unfertilized egg contains 5 X 10(5)-fold the myc RNA content of a proliferative somatic cell. The myc RNA store is evenly distributed in the oocyte and the egg. Fertilization triggers a post-transcriptional control of the gene and the RNA store is progressively degraded to a constitutive value of 10 to 30 myc RNA copies registered per gastrula embryonic cell. The 62K myc protein is accumulated late in oogenesis. This uncoupling of myc expression and cell proliferation appears as a specific developmental regulation of the myc gene, adapted to the series of rapid cell cleavages occurring after fertilization.

Both N-ras and c-myc are activated in the SHAC human stomach fibrosarcoma cell line.

A transforming N-ras gene was isolated from the SHAC human stomach fibrosarcoma cell line. A single-point mutation resulting in the substitution of histidine for glutamine at codon 61 was found in the SHAC transforming allele. The N-ras gene is overexpressed in the tumor cells and transformant cells. The N-ras p21 product was studied by immunoprecipitation and showed no alteration in mobility as compared to the normal p21 protein. The c-myc gene is amplified and overexpressed in these cells. This report gives evidence that an amplified c-myc and a mutated N-ras gene are both present in this tumor cell line and provides support for the idea that co-operation of at least 2 activated cellular oncogenes is required for carcinogenesis.

A c-ras-Ki oncogene is activated, amplified and overexpressed in a human osteosarcoma cell line.

We present a characterization of an activated oncogene which we found to be present in DNA of the OHA osteosarcoma cell line. We identify this tumor oncogene which transforms Swiss mouse 3T3-cells, with c-ras-Ki 2, one of two known members of the Kirsten ras family of human proto-oncogenes. Its structural outlines are given and we show that: 1) a single point mutation causing a substitution of valine for glycine in codon 12 was found by DNA sequencing; 2) the c-ras-Ki gene is amplified and overexpressed in the original OHA tumor cells and its transformants and 3) the gene product is an abnormal form of the p21 protein.

Interferon-induced phenotypic changes in human tumor cells relative to the effects of interferon on c-ras oncogene expression.

Three human tumor cell lines derived from an osteosarcoma (OHA cells), a bladder carcinoma (EJ cells), and a gastric sarcoma (SHAC cells) were passaged serially in the presence of human interferon-alpha (IFN-alpha) for extended periods of time. The long-term IFN-alpha treatment induced a partial reversion of OHA tumor cell phenotype as exemplified by inhibition of cell proliferation, lack of cellular overlapping in confluent cultures and marked reduction in tumorigenicity. In contrast, under the same conditions, long-term IFN treatment did not reverse but even potentiated some of the phenotypic characteristics (including tumorigenicity) of EJ and SHAC cells. In the three tumor cell lines, the transforming ability, genomic level, or expression of activated oncogenes, c-Ki-ras, c-Ha-ras, and N-ras, respectively, were unaltered with long-term IFN-alpha treatment. Our data indicate that IFN-induced phenotypic changes are not necessarily associated with changes in oncogene expression.

Evolution in the structure and distribution of 4F2-antigen from the oncofetal to the adult phenotype of human fibroblasts.

The monoclonal antibody (MAb) 4F2 defines an oncofetal antigen in human fibroblastic cells. Two-dimensional electrophoretic analysis reveals that tumor cell lines from mesenchymal tissues co-express two or more heavy-chain molecular variants of the antigen whereas the light subunit (41 kDa) is not affected. Among normal cells, only embryonic and newborn fibroblasts (from donors up to 20 days after birth) clearly co-express two distinct molecular forms of the heavy chain with MW of 85 and 75 kDa, respectively. Cells derived from 3-month-old donors express detectable amounts of the 85 kDA but only faint traces of the 75 kDa subunit, while fibroblastic cells derived from donors older than 3 months seem to express only the 85 kDa subunit. Immunofluorescence analysis performed on adherent living cells shows that, in the first months after birth, there is a gradual evolution from the oncofetal to the adult phenotype also in the cell distribution of the 4F2. This evolution is reflected by a progressive disappearance of the 4F2 antigen from the cell membrane becoming, in adult normal cells, inaccessible to anti-4F2 MAb. The existence of different molecular forms and different membrane positions of the 4F2 antigen could facilitate surveillance of morphological and structural changes in the evolution of human fibroblastic cells during the developmental process and neoplastic transformation.