The effect of transplanting donor spleen with an organ allograft.
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Animals↗
Biomedical subjects
J M Blanchard
Publications and source records attributed to J M Blanchard.
c-fos mRNA constitutive expression by mature human megakaryocytes.
By in situ hybridization with a c-fos probe, we have shown that human bone marrow megakaryocytes cultured in the presence of 20% aplastic anemia plasma constitutively express c-fos mRNA. At day 0, megakaryocytes are mostly immature and only 3% of them are labeled. The number of labeled cells reached 23% after 12 days of culture. Interleukin 3 (IL-3) and IL-6 added together at day 10 further increased this number to 31% 2 days later. Mature labeled megakaryocytes were more numerous and more strongly labeled than immature ones. These results suggest that c-fos could play a role in megakaryocytic terminal differentiation, either in the polyploidization or in the thrombopoietic function unique to these cells.
Genes, fos↗
In vivo footprints between the murine c-myc P1 and P2 promoters.
Assuming that when transcription starts at the P2 promoter of the c-myc gene sites located immediately upstream from P2 are occupied whereas in the absence of initiation they are not, the polymerase chain reaction (PCR)-based method of Mueller & Wold [(1989). Science, 246, 780-786] was used to map in vivo footprints upstream from the P2 promoter in various mouse cell lines. In cultured Friend erythroleukemic cells induced to differentiate with dimethysulfoxide (DMSO), a clear protection corresponding to ME1a2 and E2F sites was observed, consistent with in vitro band-shift and footprint data. However, in cell lines in which the gene was either silent or truncated the footprints were no longer visible. Friend c-myc transcripts decreased to a barely detectable level after 3 h of DMSO treatment. Transcription, as measured by in vitro run-on, was turned off at the level of RNA polymerase elongation rather than initiation [Mechti N., Piechaczyk, M. Blanchard, J.-M., Marty, L., Bonnieu, A., Jeanteur, Ph. & Lebler, B. (1986). Nucleic Acids Res., 24, 9653-9666]. The state of occupancy of the sites did not vary from the first hours up to 9 days of DMSO treatment, suggesting that DNA occupancy per se cannot explain premature termination, which rather would involve a more complex phenomenon.
Animals↗
Casein kinase II induces c-fos expression via the serum response element pathway and p67SRF phosphorylation in living fibroblasts.
Elevation of intracellular casein kinase II (CKII) levels through microinjection of purified CKII results in the rapid and transient induction of c-fos in quiescent rat embryo fibroblasts, and activation of quiescent cells by serum is accompanied by the nuclear relocation of endogenous CKII. The induction of c-fos by CKII is inhibited by coinjection of oligonucleotides corresponding to the sequence of the serum response element (SRE) present in the c-fos promoter, indicating that competitive displacement of positive factors from the endogenous c-fos SRE prevents c-fos induction by CKII. Furthermore, the expression of c-fos induced by either CKII injection or serum activation is also inhibited by microinjection of antibodies against the 67 kDa serum response factor (p67SRF) indicating the absolute requirement of p67SRF in this process. Finally, we show the specific phosphorylation of p67SRF in vivo following microinjection of CKII into quiescent cells. Together, these data strongly support that CKII induces c-fos expression through binding/activation of the phosphorylated p67SRF at the SRE sequence.
Animals↗
p67SRF is a constitutive nuclear protein implicated in the modulation of genes required throughout the G1 period.
Indirect immunofluorescence analysis, using antibodies directed against peptide sequences outside the DNA-binding domain of the 67-kDa serum response factor (p67SRF), revealed a punctuated nuclear staining, constant throughout the cell cycle and in all different cell lines tested. p67SRF was also tightly associated with chromatin through all stages of mitosis. Inhibition of p67SRF activity in vivo, through microinjection of anti-p67SRF antibodies, specifically suppressed DNA synthesis induced after serum addition or ras microinjection, suggesting that these antibodies were effective in preventing expression of serum response element (SRE)-regulated genes. A similar inhibition was also obtained in cells injected with oligonucleotides corresponding to the DNA binding sequence for p67SRF protein, SRE. Moreover, this inhibition of DNA synthesis by anti-p67SRF or SRE injection was still observed in cells injected during late G1, well after c-fos induction. These data imply that genes regulated by p67SRF are continuously involved in the proliferation pathway throughout G1 and that p67SRF forms an integral component of mammalian cell transcriptional control.
Amino Acid Sequence↗
C-fos and c-jun proto-oncogene expression is decreased in psoriasis: an in situ quantitative analysis.
Psoriasis is a common, sometimes severe, non-malignant skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. Because proto-oncogenes are implicated in both cell proliferation and differentiation, their expression could be modified in skin diseases such as psoriasis. The c-fos and c-jun proto-oncogenes, whose products associate to form a heterodimeric transcription factor, are among the first genes to be expressed when certain cells are stimulated to either proliferate or differentiate. Recent studies in our laboratory have shown that the c-fos proto-oncogene is highly expressed in normal human adult skin. In the present study, we used in situ hybridization with RNA to compare the expression and localization of c-fos and c-jun transcripts in 15 lesional and non-lesional psoriatic skin samples. Two clinical variants of psoriasis were studied: the most severe and chronic form or plaque-type psoriasis (N = 10) and rapidly resolutive guttate-type psoriasis (N = 5). Quantitative analysis was performed using a semi-automatic image analyzer and the "Starwise grain" software program. Our control samples included 10 normal skins and eight specimens from other benign hyperproliferative non-psoriatic skin diseases, consisting of three with inflammation (seborrheic dermatitis and atopic dermatitis), and 5 without inflammation (seborrheic keratoses). Control genes we used for in situ hybridization and RNA integrity were keratin 14, which is expressed in the epidermis and was normally expressed in all tissue analyzed, and ribosomal RNA. Our data showed that c-fos and c-jun were expressed to an equivalent extent, both spatially and quantitatively, in all specimens tested. Expression was significantly decreased in plaque-type but not in guttate-type psoriasis. It was also decreased in the three other benign inflammatory cutaneous hyperproliferative disorders, but not in the five non-inflammatory cases. These results were surprising because hyperproliferation was here associated with a decrease in proto-oncogene expression, thus suggesting that c-fos and c-jun do not play a crucial role in the control of keratinocyte proliferation in vivo. However, their reduced expression in some abnormally differentiated skins indicates that both c-fos and c-jun proto-oncogenes may play a key role in keratinocyte differentiation. Their altered expression correlated with severity of the disease and the presence of an inflammatory infiltrate. These data offer a new insight into the role and regulation of these proto-oncogenes in vivo in humans.
Chronic Disease↗
c-fos gene transcription in murine macrophages is modulated by a calcium-dependent block to elongation in intron 1.
Cultured mouse thioglycolate-elicited peritoneal macrophages exhibit a strong block to transcriptional elongation beyond the end of the c-fos gene first exon. This block is absent in freshly isolated peritoneal cells, appears slowly during culture, and does not require adherence of the cells. The extent of this block is largely responsible for the levels of c-fos mRNA in cultured macrophages, even after modulation by agents such as the tumor promoter phorbol myristate acetate and increased intracellular cyclic AMP, which also increase the activity of the c-fos promoter. When macrophages are cultured in the absence of mobilizable calcium, the block can no longer be relieved by any inducing agent. Conversely, upon calcium influxes, there is little alteration in the level of transcriptional initiation, but transcription proceeds efficiently through the entire c-fos locus. These results suggest the presence of an intragenic calcium-responsive element in the c-fos gene and illustrate its key role in the control of c-fos gene transcription.
Animals↗
Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene.
A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.
Animals↗
FK 506 enhances the beneficial effects of donor-specific blood transfusion on allograft survival in rats.
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Animals↗
Nuclear localization of c-Fos, but not v-Fos proteins, is controlled by extracellular signals.
We report here that transport of the protein product of the c-fos proto-oncogene from the cytoplasm, where it is synthesized, into the nucleus, where it operates as part of the AP-1 transcription complex, is not spontaneous but depends on the continuous stimulation of cells by serum factors. A labile protein inhibitor of transport, the effect of which is reversed by cAMP, is responsible for retention of c-Fos protein within the cytoplasm of serum-starved fibroblasts. In contrast, v-Fos proteins transduced by the murine retroviruses FBJ and FBR, which remain nuclear in the absence of serum, evade the translocation control, which therefore appears to contribute to their tumorigenic potential.
Animals↗
Demonstration in living cells of an intragenic negative regulatory element within the rodent c-fos gene.
We studied c-fos gene expression in rat fibroblasts by microinjection of regulatory DNA sequences, such as the serum response element (SRE) present in c-fos promotor, in order to compete directly with such sequences for binding of putative regulatory factors. We show that an additional fos intragenic regulatory element (FIRE) is located at the end of exon 1. When coinjected with an SRE oligonucleotide, it induced c-fos expression in quiescent cells, whereas injection of SRE sequence alone failed to do so. Moreover, injection in quiescent cells of an SRE oligonucleotide together with a p-fos-lacZ construct containing the c-fos SRE as well as an in-frame insertion of FIRE resulted in a block to beta-galactosidase expression that can be relieved by coinjection of the FIRE sequence.
Animals↗
High levels of c-fos proto-oncogene expression in normal human adult skin.
The proto-oncogene c-fos is thought to play an important role in the modulation of cell growth and differentiation. In normal tissues that have been studied to date, c-fos expression has been found to be regulated in a tissue-specific manner. Actually, little is known about its expression in normal human adult skin (NHAS). Moreover, the epidermis is a useful tissue to study the role of cellular oncogenes because keratinocytes can be observed simultaneously in their proliferative as well as differentiated state. We studied c-fos expression in NHAS using different molecular approaches which permit us to characterize and localize c-fos products within the epidermis, specifically, at the RNA level by Northern blot and in situ hybridization, and at the protein level by immunofluorescence and Western blotting. Here, we show that both c-fos mRNA and protein are present at high levels in NHAS. These results contrast with the low level of c-fos expression reported for most human adult tissues. Furthermore, c-fos expression is visible throughout the epidermal layers indicating that it is not restricted to proliferating basal cells. The epidermis, therefore, represents the first human adult tissue where c-fos is expressed at high levels in vivo and provides an interesting model to further elucidate the role of this proto-oncogene in normal and pathologic conditions.
Blotting, Northern↗
The muridae glyceraldehyde-3-phosphate dehydrogenase family.
Although only one gene is known to be functional, numerous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences are scattered throughout Mus musculus and Rattus rattus genomes. In this report we show that: (1) GAPDH pseudogenes are repeated to comparable extents, at least 400 copies, in 12 other Muridae species; (2) the complete, or nearly so, sequence of GAPDH messenger RNA is amplified, and a high proportion, if not all of these copies, are intronless; (3) GAPDH pseudogenes are preferentially located in heavily methylated and DNAse I-insensitive regions of chromatin; and (4) the presence of atypical GAPDH-related mRNAs in different cellular contexts raises the possibility that more than one GAPDH gene is transcribed.
Animals↗
c-myc and c-fos gene regulation during mouse liver regeneration.
We have examined the expression of c-myc and c-fos proto-oncogenes in regenerating mouse liver, in order to analyse the relative contributions of transcriptional and post-transcriptional regulations in vivo. We show that c-myc and c-fos transcription is induced after partial hepatectomy, and involves common mechanisms. A strong block to transcriptional elongation exists in normal liver, within the first exon of c-myc gene. This block is only slightly relieved during liver regeneration, although transcriptional initiation is transiently increased (4-6 fold). In contrast, transcription initiation of c-fos is induced while the transcriptional block within the first exon of the gene is almost completely abolished. The steady-state levels of both transcripts increased to high levels (50-100 fold) within 2-6 h after partial hepatectomy, and were maintained for at least 40 h in the case of c-myc. We conclude that post-transcriptional control mechanisms are largely responsible for the dramatic induction of c-myc mRNA in regenerating liver, while c-fos mRNA accumulation is the result of both an increased initiation and a relief of a transcriptional block to elongation. Induction of both genes in vivo with cycloheximide argues in favor of negative trans-acting proteins which regulate initiation and elongation of transcription.
Animals↗
In situ and in vitro evidence for intragenic binding of nuclear factors at the murine c-myc locus.
A block to transcriptional elongation within the c-myc proto-oncogene has been previously observed in a large number of different mouse and human cell types and its release is a potentially important element in the pathogenesis of some malignancies. We show here that the chromatin around the mouse c-myc exon 1-intron 1 boundary is differentially accessible to restriction enzymes in purified nuclei. Using a combination of in situ exonuclease III protection assay with in vitro footprints and gel band shifts, we have shown the existence of a stable nucleoprotein complex in this same region in mouse erythroleukemia cell nuclei. This situation is not peculiar to these cells and we have shown that the accessibility of the two BglII sites present at the beginning of intron 1 seems to depend not only upon the transcriptional state, but also upon the structural integrity of the gene.
Animals↗
Role of RNA structures in c-myc and c-fos gene regulations.
Proto-oncogenes c-myc and c-fos are subjected to a complex set of controls operating both at the transcriptional and post-transcriptional levels. We report here that: (i) antisense transcription occurs at the murine c-myc locus. However, its biological significance remains to be established; (ii) transcription of both genes is regulated in various situations by a block to elongation of nascent RNA chains. In the case of c-myc, the blockade involves a RNA structure whose nature remains unknown; (iii) elements responsible for the high degree of instability of c-myc and c-fos mRNAs reside in their 3' non-coding regions. A U-rich region, reminiscent of that present in the granulocyte-monocyte colony-stimulating factor mRNA destabilizer, is likely to be involved in the rapid degradation of c-fos mRNA; (iv) exon 1 substitution by intron 1-derived sequences lessens or negates the effect of the 3' destabilizer in abnormal c-myc RNAs from Burkitt's lymphomas and mouse plasmacytomas.
Animals↗
Inhibition of cytochrome P-450p (P450IIIA1) gene expression during liver regeneration from two-thirds hepatectomy in the rat.
Regenerating liver from partial hepatectomy (HPX) is known to exhibit a strong and transient deficiency in both spectrally detectable microsomal cytochrome P-450 (P-450) and related monooxygenase activities. Male Wistar rats (250-300 g) were HPX or sham operated and liver was excised at different times after operation. The time course of accumulation of five different forms of P-450 (including P-450b/e, P-450c, P-450d, P-450p and P-450UT-A) was determined in the regenerating liver, by Western blots developed with specific antibodies. With the exception of P-450c, whose level was not affected, the accumulation of other forms strongly decreased during the first 24 hr after HPX. For P-450b/e and P-450d, 80% of initial level was restored at 96 hr, whereas for P-450p and P-450UT-A, two major forms in control rat liver, the accumulation was only 20-25% of the initial, 1 week after HPX. No significant decrease was observed in sham operated animals. Plasmid pDex 12 containing a cDNA insert coding for P-450p was used to further investigate the effects of HPX on P-450p mRNA level and gene transcription. Northern blot analysis of RNA from regenerating liver (cDNA insert of pDex 12 being used as a probe) demonstrated that P-450p mRNA level decreased strongly to a minimum 12 hr after operation. This was correlated with a strong and transient decrease in P-450p gene transcription determined from nuclear run on experiments, the time course of which, however, did not account for the early decrease in mRNA level. We conclude that P-450p deficiency in the regenerating liver results from a combination of transient inhibition of gene transcription and early increase of mRNA degradation. Time course and amplitude of the decrease in P-450 UT-A accumulation suggest an inhibition of gene transcription as observed with P-450p.
Animals↗
Interferons and oncogenes in the control of cell growth and differentiation: working hypothesis and experimental facts.
This short review summarizes available evidence for (i) growth regulatory properties of exogenous as well as recently described autocrine IFNs, (ii) down-regulation of cellular oncogene expression with emphasis on c-myc and (iii) the possible involvement of the IFN-regulated 2-5A pathway at these levels. Initially described as a part of the IFN-induced antiviral mechanism, this double-stranded RNA-activated pathway leads to the preferential degradation of viral mRNAs in IFN-treated virus-infected cells probably through localized activation at the site of virus replication. Such mechanisms could be involved in the regulation of the stability of rapidly turning over mRNAs as for instance c-myc mRNA in IFN-treated cells. Whatever the elegance of the concept, however, experimental evidence is essentially circumstantial; tools developed in our group to strengthen the demonstration are briefly described.
Animals↗