Philippe Jeanteur

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.

The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker

Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.

Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells: Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs☆

Abstract U 1 snRNP † isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U 1 molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U 1 core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle. When a less drastically purified U 1 snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected. When a mixture of U 1 , U 2 , U 4 , U 5 and U 6 snRNPs, which contains the same four/five polypeptides as U 1 snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U 2 , U 4 and U 5 were found in addition to those derived from U 1 . No fragment derived from U 6 was found. In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U) n G with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U 2 , where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U 6 snRNA, it does not contain the sequence A(U) n G which correlates well with the fact that no U 6 snRNA fragment seems to resist micrococcal nuclease digestion.

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.

Cathepsin D assay in primary breast cancer and lymph nodes: Relationship with c-myc, c-erb-B-2 and int-2 oncogene amplification and node invasiveness

In breast cancer, axillary lymph node invasiveness is the major prognostic factor in predicting relapse and metastasis. Nevertheless, since 30% of node-negative tumors also relapse, it is necessary to develop other independent prognostic factors. Oncogene amplification and the level of cathepsin D (cath-D), an acidic lysosomal protease produced and secreted in excess by breast cancer cells, have been proposed as additional prognostic factors. We have compared the cytosolic cath-D level and the amplification of three oncogenes: c-myc, neu-erb-B-2 and int-2 in 140 primary breast carcinomas and 64 axillary lymph nodes collected in 1987 and 1988 at the Cancer Center of Montpellier (Centre Paul Lamarque). None of the patients had previously received hormonal or chemotherapy. The cath-D concentration was measured with an immunoradiometric assay using monoclonal antibodies. DNA purified from the same samples was analyzed by a standard Southern blotting technique to estimate oncogene amplification. No correlation was found between the level of cath-D in the tumor and node invasiveness. Using a cut-off level of 60 pmol/mg protein, the status of cath-D was not correlated with neu-erb-B-2 and int-2 amplification and only correlated with c-myc amplification (P = 0.011). Both c-myc and cath-D are associated with cell proliferation, induced by estrogens in ER+ breast cancer, and constitutively produced in ER- breast cancer. The level of cath-D was significantly higher in the invaded lymph nodes (P = 0.04) than in the histologically non-invaded ones. Nevertheless, some non-invaded lymph nodes contained a high level of cath-D, as confirmed by immunoperoxidase staining. In conclusion, in breast cancer, a high cytosolic cath-D concentration is more frequent in tumors with c-myc amplification but is dissociated from neu-erb-B-2 or int-2 amplification, suggesting that the determination of these three markers will have an additional prognostic value.

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.

Small-Molecule Inhibition of HIV pre-mRNA Splicing as a Novel Antiretroviral Therapy to Overcome Drug Resistance

The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16) that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell–tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

Characterization of nuclear RNP particles from Hela cells. Analysis of protein and RNA constituents. Presence of poly (A).

Summary The present work reports on the occurence in HeLa cell nuclei of ribonucleoprotein particles very similar to those already described in various tissues. Most of these particles have sedimentation coefficients arround 30–40S (monoparticles) although much larger structures, up to 200S, have been observed (polyparticles). These particles have the characteristic low density in CsCl (1.39–1.41 g/cm3). Their RNA moiety exhibits the high A-U content and the absence of pseudouridylic acid characteristic of HnRNA. The presence of poly(A) in the RNA extracted from the particles has been established. SDS acrylamide gel electrophoresis of their proteins shows a complex pattern with sizes ranging from 35,000 to 150,000 in contradiction with the existence of a unique protein (informofer).

Variation of bcl-2 expression in breast ducts and lobules in relation to plasma progesterone levels: overexpression and absence of variation in fibroadenomas

Some women with benign breast disease eventually develop breast cancer. The mammary gland undergoes tissue remodelling according to hormonal influences, involving a balance between quiescence, proliferation, and mechanisms of cell death. Proliferation and/or apoptotic events could therefore be investigated to help understand the mechanisms of benign lesion formation and identify mastopathies with a poor prognosis. bcl‐2 expression was analysed by immunohistochemistry in 75 benign mastopathies. Protein levels were quantitated with an image analyser in various epithelial structures on frozen sections, including adenoses, fibroadenomas, ductal epithelial hyperplasias, cysts, and apparently normal surrounding lobules and ducts. bcl‐2 levels were equivalent in apparently normal lobules and ducts, as well as in cysts and ductal hyperplasias. bcl‐2 staining was significantly higher in fibroadenomas, known to be of lobular origin [mean=10·1, quantitative immunochemistry score (QIC) arbitrary units (AU), n=19], than in normal lobules (mean=5·1 AU, n=43, P=7×10−5). bcl‐2 levels in normal lobules and ducts varied according to the menstrual cycle, being higher during the follicular than the luteal phase (P=1·8×10−2 and P=1·7×10−2 respectively). This was further supported by a statistical link (P=5×10−3) between high levels of circulating progesterone and weak bcl‐2 staining in lobules and ducts. This progesterone‐dependent variation was absent in fibroadenomas. No statistical correlation was found between bcl‐2 expression and circulating levels of oestradiol, and follicle‐stimulating or luteotrophic hormones. Although these are only preliminary results, they suggest an influence of progesterone on bcl‐2 expression which might be lost in fibroadenomas. A hypothesis is proposed concerning the potential involvement of altered regulation of the apoptotic process in the formation of such benign lesions.

c-myc gene regulation still holds its secret

Abstract The c- myc oncogene is proving toe extremely complex, both in structure and in its regulation at the transcriptional and post-transcriptional levels. The significance of the complex regulatory mechanisms both for the normal function of the gene and for its aberrant function as an oncogene remain unknown.