Growth-regulated expression of rhoG, a new member of the ras homolog gene family.

Cellular transition from the resting state to DNA synthesis involves master switches genes encoding transcriptional factors (e.g., fos, jun, and egr genes), whose targets remain to be fully characterized. To isolate coding sequences specifically accumulated in late G1, a differential screening was performed on a cDNA library prepared from hamster lung fibroblasts stimulated for 5 h with serum. One of the positive clones which displayed a sevenfold induction, turned out to code for a protein sharing homology to Ras-like products. Cloning and sequence analysis of the human homolog revealed that this putative new small GTPase, referred to as rhoG, is more closely related to the rac, CDC42, and TC10 members of the rho (ras homolog) gene family and might have diverged very early during evolution. rhoG mRNA accumulates in proportion to the mitogenic strength of various purified growth factors used for the stimulation, as a consequence of transcriptional activation. G1-specific RNA accumulation is impaired upon addition of antimitogenic cyclic AMP and is enhanced when protein synthesis is inhibited, mainly as a result of RNA stabilization. rhoG mRNA expression is observed in a wide variety of human organs but reaches a particularly high level in lung and placental tissues.

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.

Mammalian U6 small nuclear RNA undergoes 3' end modifications within the spliceosome.

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.

Mg2+ induces a sharp and reversible transition in U1 and U2 small nuclear ribonucleoprotein configurations.

When U1 and U2 small nuclear ribonucleoproteins (snRNPs) purified by a procedure which preserves their immunoprecipitability by autoimmune antibodies (Hinterberger et al., J. Biol. Chem. 258:2604-2613, 1983), were submitted to extensive digestion with micrococcal nuclease, we found that their degradation pattern was sharply dependent upon magnesium concentration, indicating that they undergo a profound structural modification. At low Mg2+ (less than or equal to 5 mM), both particles only exhibit a core-resistant structure previously identified as being common to all but U6 snRNAs (Liautard et al., J. Mol. Biol. 162: 623-643, 1982). At high Mg2+ (greater than or equal to 7 mM), U1 and U2 snRNPs behave differently from one another. In U1 snRNP, most U1 snRNA sequence is protected, except for the 10 5'-terminal nucleotides presumably involved in splicing and a short sequence between nucleotides 102 and 108. Another region spanning nucleotides 60 to 79 is only weakly protected. This structural modification was demonstrated to be reversible. In U2 snRNP, the U2 snRNA sequence remains exposed in its 5' part up to nucleotide 92, and the 3'-terminal hairpin located outside the core structure becomes protected.

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.

p53 mutations occur in aggressive breast cancer.

Using a polymerase chain reaction-single strand conformation polymorphism approach we analyzed 96 human primary breast tumors for the presence of mutations in exons 2, 5, 6, 7, 8, and 9 of the p53 gene. These exons have been shown to comprise highly conserved sequences and the portion including exons 5 through 9 is believed to be the target for over 90% of the acquired mutations in human cancer. Eighteen tumors of the 96 (18.7%) tested showed reproducibly a variant band indicative of a mutation. Most (15 tumors) of the mutations were single nucleotide substitutions and G:C to A:T transitions were prevalent (6 tumors), G:C to T:A transversions came next (4 tumors), and guanines were always on the nontranscribed strand. Concomitant loss of the wild type allele and mutation of the other copy was observed in only 3 of 18 mutated cases; this is consistent with the heterogeneous cellular composition of breast tumors. Furthermore p53 mutations were correlated to estrogen and/or progesterone receptor negative tumors, thus indicating their relationships to aggressive breast cancer. No association could be observed with DNA amplification events in these tumors.

Requirements for c-fos mRNA down regulation in growth stimulated murine cells.

The fos proto-oncogene is rapidly and transiently expressed in resting cells exposed to growth stimulation. This gene is down-regulated at least at two levels: transcriptional repression and mRNA degradation. To determine the sequences and the structures involved in mRNA instability, we analyzed in mouse Ltk- cells various fos/beta-globin constructs for their transcriptional activity and the half-lives of the corresponding RNAs. In these cells, rabbit beta-globin genes under the control of a 500 bp fos SRE (serum responsive element)/promoter region are transiently transcribed within 30 min after stimulation. Analysis of the decay kinetics of RNA originating from these constructs led to the following conclusions with respect to the nature of c-fos destabilizer elements: (i) 100 bases from c-fos 3' untranslated region are able to confer instability when inserted into a normally stable beta-globin RNA; (ii) however, the degradation is more rapid when the complete untranslated region is inserted; (iii) rapid mRNA breakdown requires more determinants than two AUUUA motives and is associated with a reduction in size, presumably due to a poly(A) shortening; (iv) remarkably, c-fos destabilizing sequences remain active even when part of the coding sequence.

p53 mutations in ovarian cancer: a late event?

Using a combination of polymerase chain reaction and single-strand conformation polymorphism techniques we analyzed 34 ovarian cancer samples (30 primary tumors and four matched metastases) for the presence of mutations in exons 5, 6, 7, 8 and 9 of the p53 gene. Mutations in this portion of the gene are known to lead to the loss of the oncosuppressive potential of p53. Thirty-six percent (11/30) of the ovarian carcinomas tested presented a mutated p53 allele. Mutations were clustered in exons 5 and 7 to the exclusion of the other exons screened. Most mutations (10/11) were point mutations, but no preferential pattern of nucleotide substitution could be observed. In three tumors the mutation of one allele was concomitant with the loss of the wild-type counterpart. Another sample presented both alleles independently mutated. These observations are in agreement with the recessive nature of the p53 mutation. However, analysis of tissue sections from two tumors showed that the portion composed of 100% cancer cells could hold both the mutated and the wild-type form. Moreover analysis of serial sections gave evidence of a heterogeneous cellular content in one of these tumors, suggesting that p53 mutations may, in some cases, occur late during ovarian cancer evolution. It is, moreover, noticeable that, in matched sets of primary tumors and metastases, the same mutation was observed in both tumor samples. Therefore, even as a late event, p53 mutation occurs before metastatic spread.

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.

Nucleotide sequence polymorphism in a hotspot mutation region of the p53 gene.

By screening for mutations in the p53 coding sequence by means of single-strand conformation polymorphism (SSCP) in a series of breast tumors we detected a novel polymorphism. This change in the SSCP pattern was detected in 6.2% of the tumor DNAs analysed and implied an A to G substitution at the last base of codon 213, thus representing a neutral change. First suspecting a somatic mutation we confirmed its presence in matched sets of DNAs from normal tissues. Extending our study to a series of 60 ovarian carcinomas and 70 healthy blood donors we noticed that this polymorphism represented only 3% and 2.6% respectively. We wondered if the difference in frequency in the breast cancer population might not be related to familial breast cancer and analysed 26 DNAs from patients showing predisposition to the disease. Two patients presented this polymorphism and one corresponding kindred was analysed, revealing a mendelian mode of transmission but no correlation with the cancer phenotype.

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.

Characterization of late response genes sequentially expressed during renewed growth of fibroblastic cells.

Many proto-oncogenes are rapidly and transiently activated during the early stages of the cellular transition from a resting G0 state to the DNA synthesis (S) phase. To get better understanding of the gene complexity involved at later stages, we isolated, by cDNA cloning, and identified 17 genes that are activated sequentially during the period of time from proto-oncogene expression to the onset of DNA synthesis in the hamster CCL39 fibroblastic cell line. When protein synthesis is inhibited, induced expression of these genes is unaffected for 10 of them, enhanced for four, in a fashion similar to the immediate-early response genes, and inhibited for three, as observed for delayed early-response genes. In addition to rhoG, a new member of the ras homolog gene family (Vincent et al., 1992), cDNA sequencing indicated that six of them correspond to cytoskeletal proteins (alpha-tubulin, vascular alpha-actin and skeletal gamma-actin), extracellular matrix protein (thrombospondin), secreted protease (plasminogen activator inhibitor-1) and energy-linked transporter (mitochondrial proton/phosphate symporter). This overall survey shows that numerous differentially regulated gene activations are associated with the cell cycle progression, and suggests that proteins involved in cellular reshaping participate actively in the control of cellular growth.

AUUUA motifs are dispensable for rapid degradation of the mouse c-myc RNA.

Sequence determinants responsible for c-myc RNA rapid turn-over are localized within the 3' non-coding region which is mainly characterized by the presence of two polyadenylation signals and a high content in A and U. Although the AUUUA/UUAUUUA motif is commonly thought to specify a whole class of unstable RNAs coding for various onco-proteins and cytokines, site-directed mutagenesis showed that both of the two such sequences found in the mouse c-myc RNA are dispensable for rapid RNA degradation. Although less efficient than the whole 3' non-coding region, the last 50 nucleotides of c-myc RNA, mainly made up of U and A and devoid of AUUUA/UUAUUUA motif, are sufficient to confer instability to the coding sequence.

p53 gene mutations in human epithelial skin cancers.

In the present study we analysed 38 epithelial skin cancers, 19 basal cell carcinomas (BCCs), 13 squamous cell carcinomas (SCCs) and six Bowen diseases (BwDs), using a combination of polymerase chain reaction (PCR) and single-stranded conformation polymorphism (SSCP) techniques for the presence of p53 and RAS gene mutations. Whereas 48% (9/19) of the BCCs tested presented a mutated p53 gene, the frequency was lower (15%, 2/13) in our series of SCCs and negative in the BwDs. Nine of the 11 characterized mutations were single-nucleotide substitutions and, interestingly, seven of these involved CC dimers, where a C was changed into a T or a G (three C-->T transitions and four C-->G transversions). This mutational pattern, added to the fact that all the mutated tumors occurred at sun-exposed body sites, implicates UV light in their genesis. Furthermore, we observed two internal deletions of 6 and 24 bp whose flanking sequences contained two or three Cs on either strand. In addition to molecular detection, we searched for p53 protein accumulation, by immunocytochemical staining, in a subset of 23 epithelial skin tumors (nine bearing a mutation, 14 which scored negative in our assay). Three commercially available anti-p53 antibodies (PAb CM1, mAbs DO7 and 1801) were used, and 3/23 (all showing a mutated p53 gene) presented specific nuclear staining. In contrast to other reported data we could not detect any activating RAS gene mutation in our series of human skin cancers.

p53 and RAS gene mutations in multiple myeloma.

We analysed genomic DNA from 30 patients with multiple myeloma (MM), searching for alterations in the p53 and RAS genes by a combination of polymerase chain reaction and single-strand conformation polymorphism techniques. Mutations in the p53 gene were observed in 20% (6 out of 30) of the patients, and were located in conserved sequence blocks within exons 5 and 7. These were single-nucleotide substitutions and consisted predominantly (4/6) of G:C to A:T transitions. Of the six patients with a mutated p53 gene, four were in the terminal phase of the disease. RAS gene mutations were found more frequently since they occurred in 47% (14 out of 30) of the patients. Mutations consisted of single-nucleotide substitutions, located in codons 12, 13 and 61 of either K- or N-RAS, to the exclusion of H-RAS. Moreover, one patient bore two simultaneous mutations, affecting simultaneously the K- and the N-RAS genes. RAS gene mutations were more frequently observed in patients with fulminating disease (10/15, 67%) than in patients with less aggressive forms of the disease (4/15, 26%). We also analysed genomic DNAs from 10 human myeloma cell lines, of which two bore mutations affecting codon 12 of the K-RAS gene, and one codon 12 of the N-RAS gene. The first two cell lines were obtained from freshly explanted tumor cells in which we observed identical mutations. Results presented here show that activating mutations in the RAS genes are, in MM, more frequent than those affecting the p53 gene and suggest that both events are related to terminal phases of the disease.

Proto-oncogene amplification and human breast tumor phenotype.

Amplification of c-myc, c-erbB-2, hst and int-2 proto-oncogenes was investigated in two independently collected breast tumor series comprising 292 carcinomas. Differences in the frequencies of amplification could be observed between these two series for c-myc (9.3% vs. 20.8%) and hst/int-2 (21.5% vs. 15.6%) whereas similar values were found for c-erbB-2 (22.5% vs. 20.3%). Statistical correlations between amplification and disease parameters were also dependent on population sampling. Therefore we performed our statistical analysis on the pooled populations and focused on the 219 primary breast carcinomas from patients without therapy prior to surgery. Amplification of c-erbB-2 was strongly correlated to the absence of either estrogen (ER-, P = 0.003) or progesterone (PR-, P = 0.004) receptors. An amplified c-myc was significantly associated with PR- (P = 0.005) and was prevalent in high grade tumors. On the contrary, hst/int-2 amplification was correlated to PR+ tumors (P = 0.01) and was more frequent in ER+ and low grade tumors, and was also correlated with lymph node involvement (P = 0.04). Our data suggest that amplification of each of these proto-oncogenes could be representative of a particular subset of breast tumors. Therefore, proto-oncogene amplification may be helpful in characterizing new biological subclasses in human breast cancer.

BCL-1 participates in the 11q13 amplification found in breast cancer.

In an attempt to probe the significance of HST and INT-2 gene amplification in human breast carcinomas, we have surveyed the amplification status of five molecular markers located on the long arm of chromosome 11 (BCL-1, HST, INT-2 & SEA on 11q13, and ETS-1 on 11q23) in a population of 297 mammary tumors. ETS-1 was rarely amplified and always independently from the other proto-oncogenes. Concerning band q13: (i) 50 tumors (approximately 17%) were co-amplified for BCL-1, HST & INT-2; (ii) in 3 cases, amplification extended to the SEA gene; (iii) in 6 carcinomas, BCL-1 was the only amplified marker. The fact that we never observed amplification of HST & INT-2 independently of BCL-1, which in turn can be amplified solely, suggests the presence, between HST/INT-2 and BCL-1, of a genetic element which could be important in the development of a subset of mammary tumors.

BEK and FLG, two receptors to members of the FGF family, are amplified in subsets of human breast cancers.

Tumor DNA samples from 387 breast carcinomas have been investigated for amplification of BEK and FLG genes, both of which have been shown to code for cell surface receptors to FGFs. BEK and FLG were found amplified in 11.5 and 12.7% of breast tumors respectively. Statistical analysis, performed on the subset of 297 primary cancers without presurgical therapy, showed for BEK a trend of preferential amplification in patients above 50 years (P = 0.055), whereas amplification of FLG could significantly be correlated with nodal involvement (P = 0.032) and seemed prevalent in steroid hormones receptor positive tumors. Since the same tumors were previously analysed for the amplification of MYC, ERBB2 and HST/INT2/BCL1 possible associations with BEK and FLG amplifications were looked for. BEK was found significantly correlated with MYC and FLG with HST/INT2/BLC1. The amplification of these two FGF receptor genes may therefore represent additional steps in the molecular phenotyping of breast cancer.

Protein methylation in animal cells. I. Purification and properties of S-adenosyl-L-methionine:protein (arginine) N-methyltransferase from Krebs II ascites cells.

1. A protein methylase which specifically transfers methyl groups from S-adenosyl-L-methionine to arginine residues of histones has been substantially purified from Krebs II ascites cells. The purified enzyme was obtained free of contamination by other protein methyl transferases specific for carboxyl and lysine residues. This latter activity copurified with the present enzyme until advanced stages of purification. 2. The purified enzyme does not require any divalent cation for maximum activity. It is inhibited by ionic strength, N-ethylmaleimide and S-adenosyl-L-homocysteine. It has an apparent molecular weight on gel filtration of approx. 5 . 10(5). A Km value for S-adenosyl-L-methionine of 2.5 . 10(-6) M was determined, while the dissociation constant Ki for S-adenosyl-L-homocysteine, which acts as a competitor, was 1.4 . 10(-6) M.

Protein methylation in animal cells. II. Inhibition of S-adenosyl-L-methionine:protein(arginine) N-methyltransferase by analogs of S-adenosyl-L-homocysteine.

1. Protein methylase I (S-adenosyl-L-methionine: protein (arginine) N-methyltransferase, EC 2.1.1.23) has recently been purified in our laboratory from Krebs II ascites cells (Casellas, P. and Jeanteur, P. (1978) Biochim. Biophys. Acta 519, 243--254). In order to probe its binding site for S-adenosyl-L-methionine, three series of compounds deriving from the most potent competitive inhibitor, S-adenosyl-L-homocysteine, by specific alterations in each of the three regions of the molecule (amino acid side chain, ribose and adenine) have been tested for inhibitor activity. A competitive type of inhibition was assumed for all of them and demonstrated for five representative ones. The contribution of each of these regions to the binding could therefore be established as follows: (i) Any modification of the side chain results in a drop in affinity of about two orders of magnitude. Adenosine itself remained significantly inhibitory thereby demonstrating that the presence of a side chain was not critical, although important. (ii) The ribose moiety appears to be an essential part of the molecule as the loss of either 2'- or 3'-hydroxyls or their change to arabino configuration resulted in a nearly complete loss of activity. (iii) The amino group at position 6 and the nitrogen atom at position 7 of the adenine ring also play a crucial role although some substitutions can be tolerated. 2. S-Isobutyladenosine was shown to specifically inhibit the methylation of arginine residues as compared to lysine.