[From fundamental research to translational research in oncology]. / De la recherche fondamentale à la recherche translationnelle en oncologie.

The present article emphasizes the crucial importance of fundamental research and translational research in oncology. These two approaches are complementary and open new perspectives for future anti-cancer therapies in clinical practice.

Cet article insiste sur l'importance de la recherche fondamentale et de la recherche translationnelle en oncologie, et sur la complémentarité de ces deux approches qui ouvrent de nouvelles perspectives thérapeutiques en clinique.

Evolution of microRNA expression during human bronchial squamous carcinogenesis.

MicroRNAs, negative post-transcriptional regulators of gene expression, are involved in cancer. Their role in early bronchial carcinogenesis was analysed in 60 biopsies obtained by fluorescence bronchoscopy (six per stage: normal tissue of nonsmokers, normal normofluorescent and hypofluorescent bronchial tissue of smokers, hyperplasia, metaplasia, mild, moderate and severe dysplasia, in situ carcinoma and invasive squamous cell carcinoma (SQCC)). In total, 69 microRNAs were found to be differentially expressed in the course of bronchial carcinogenesis. Among them, some microRNAs showed a linear evolution of their expression level, such as miR-32 and miR-34c, whose expression progressively decreased from normal bronchial tissues of nonsmokers to SQCC. Others behaved differently at successive stages, such as miR-142-3p or miR-9, or are only altered from a specific stage, such as miR-199a or miR-139. MicroRNAs globally followed a two-step evolution, first decreasing (a reverse of their increase during embryogenesis) during the earliest morphological modifications of bronchial epithelium, and thereafter increasing at later stages of lung carcinogenesis. Moreover, microRNA expression was very efficient for the prediction of the histological classification between low- and high-grade lesions and between in situ and invasive carcinoma. The present data show, for the first time, that microRNAs are involved in bronchial carcinogenesis from the very early steps of this process and, thus, could provide tools for early detection of lung cancer.

Trans activation of the bovine leukemia virus long terminal repeat in BLV-infected cells.

The transcription initiation signals for retroviruses lie within the long terminal repeat (LTR) sequences that flank the integrated provirus. This study shows that factors present in cells infected with bovine leukemia virus (BLV) mediate transcriptional trans activation of the BLV LTR. This phenomenon is similar to that reported for the human T-cell leukemia virus LTR and establishes both structural and functional criteria for inclusion of BLV and human T-cell leukemia viruses in the same family of transforming retroviruses.

Replicative and cytopathic potential of HTLV-III/LAV with sor gene deletions.

The genome of the human T-lymphotropic virus type III (HTLV-III/LAV) has the potential to encode at least three polypeptides in addition to those encoded by the gag, pol, and env genes. In this study, the product of the sor (short open reading frame) region, which overlaps the 3' end of the pol gene, was found to be a protein with a molecular weight of 23,000. An assay was developed for testing the ability of cloned HTLV-III proviruses to produce viruses cytopathic for T4+ lymphocytes. In the cell line used, C8166, neither the HTLV-III sor gene product nor the complete 3'-orf gene product were necessary for the replication or cytopathic effects of the HTLV-III.

Bovine leukemia virus-related antigens in lymphocyte cultures infected with AIDS-associated viruses.

An earlier finding that lymphocytes from African patients with the acquired immune deficiency syndrome (AIDS) react with rabbit antiserum to purified antigens of bovine leukemia virus (BLV) prompted a study of the possible cross-reactions between a BLV-infected ovine cell line and human lymphocytes inoculated with a strain of lymphadenopathy syndrome-associated virus (LAV). A solid-phase radioimmunoassay was used to detect antigenic markers of the retroviruses. Crude extracts from short-term cultures of lymphocytes infected with LAV bound rabbit antisera to the LAV glycoprotein gp13 (molecular weight 13,000) and the BLV proteins p24 and gp51, but did not bind antibodies to the p24 of human T-cell leukemia virus type I (HTLV-I). Antiserum to LAV gp13 reacted with an ovine cell line producing BLV but also weakly with virus-free ovine cells. Lymphocyte cultures from four African patients with AIDS expressed BLV-related antigens within 6 to 10 days of culture, at the moment when particle-bound reverse transcriptase was produced. BLV-related antigens were induced in lymphocyte cultures from healthy individuals by addition of filtered supernatant or irradiated cells of the original culture. The antisera to BLV used in this study may prove useful for the detection of AIDS-associated viruses in short-term cultures of lymphocytes from AIDS patients or their contacts.

The role of NPM, p14arf and MDM2 in precursors of bronchial squamous cell carcinoma.

Murine double minute clone 2 (MDM2), p14 alternate reading frame (p14arf), and nucleophosmin (NPM) regulate p53 activity. A total of 200 biopsies, including normal bronchial, pre-invasive and invasive tissues, were examined for changes in NPM, p14arf, MDM2 and p53 expression patterns by immunohistochemistry and immunofluorescence with confocal microscopy. NPM and p14arf displayed a diffuse nuclear staining in most normal bronchial tissue. The fraction of biopsies displaying an increased MDM2 staining or a nucleolar relocalisation of NPM increased at mild and moderate dysplasia, respectively. Two different modifications occurred in p14arf expression, i.e. its loss or its nucleolar relocalisation, both increasing at severe dysplasia and both being associated with high MDM2 expression. In addition, the nucleolar relocalisation of p14arf was associated with that of NPM. Immunofluorescence staining indicated that NPM and p14arf either co-localised in the nucleoplasm or in the nucleoli, before and as a result of severe dysplasia, respectively. MDM2 was not detected in the nucleoli. Thus, changes occur in murine double minute clone 2, p14 alternate reading frame and nucleophosmin level of expression and/or cellular distribution during early steps of lung carcinogenesis. Their relative localisation as determined by immunofluorescence, supports the hypothesis that p14 alternate reading frame nucleolar relocalisation impairs p14 alternate reading frame-murine double minute clone 2 complex formation and that nucleophosmin might sequester p14 alternate reading frame. The demonstration of this hypothesis requires further functional studies.

HTLV-I infection of WE17/10 CD4+ cell line leads to progressive alteration of Ca2+ influx that eventually results in loss of CD7 expression and activation of an antiapoptotic pathway involving AKT and BAD which paves the way for malignant transformation.

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy slowly emerging from human T-cell leukemia virus type 1 (HTLV-I)-infected mature CD4(+) T-cells. To characterize the molecular modifications induced by HTLV-I infection, we compared HTLV-I-infected WE17/10 cells with control cells, using micro-arrays. Many calcium-related genes were progressively downmodulated over a period of 2 years. Infected cells acquired a profound decrease of intracellular calcium levels in response to ionomycin, timely correlated with decreased CD7 expression. Focusing on apoptosis-related genes and their relationship with CD7, we observed an underexpression of most antiapoptotic genes. Western blotting revealed increasing Akt and Bad phosphorylation, timely correlated with CD7 loss. This was shown to be phosphatidylinositol 3-kinase (PI3K)-dependent. Activation of PI3K/Akt induced resistance to the apoptotic effect of interleukin-2 deprivation. We thus propose the following model: HTLV-I infection induces a progressive decrease in CD3 genes expression, which eventually abrogates CD3 expression; loss of CD3 is known to perturb calcium transport. This perturbation correlates with loss of CD7 expression and induction of Akt and Bad phosphorylation via activation of PI3K. The activation of the Akt/Bad pathway generates a progressive resistance to apoptosis, at a time HTLV-I genes expression is silenced, thus avoiding immune surveillance. This could be a major event in the process of the malignant transformation into ATLL.

Differential transcriptional activation by human T-cell leukemia virus type 1 Tax mutants is mediated by distinct interactions with CREB binding protein and p300.

The human T-cell leukemia virus type 1 Tax protein transforms human T lymphocytes, which can lead to the development of adult T-cell leukemia. Tax transformation is related to its ability to activate gene expression via the ATF/CREB and the NF-kappaB pathways. Transcriptional activation of these pathways is mediated by the actions of the related coactivators CREB binding protein (CBP) and p300. In this study, immunocytochemistry and confocal microscopy were used to localize CBP and p300 in cells expressing wild-type Tax or Tax mutants that are able to selectively activate gene expression from either the NF-kappaB or ATF/CREB pathway. Wild-type Tax colocalized with both CBP and p300 in nuclear bodies which also contained ATF-1 and the RelA subunit of NF-kappaB. However, a Tax mutant that selectively activates gene expression from only the ATF/CREB pathway colocalized with CBP but not p300, while a Tax mutant that selectively activates gene expression from only the NF-kappaB pathway colocalized with p300 but not CBP. In vitro and in vivo protein interaction studies indicated that the integrity of two independent domains of Tax delineated by these mutants was involved in the direct interaction of Tax with either CBP or p300. These studies are consistent with a model in which activation of either the NF-kappaB or the ATF/CREB pathway by specific Tax mutants is mediated by distinct interactions with related coactivator proteins.

Active versus passive anti-cytokine antibody therapy against cytokine-associated chronic diseases.

Current therapeutic vaccine trials in major chronic diseases including AIDS, cancer, allergy and autoimmunity, target antigenic pathogens but not the pathogenic stromal cytokines which can be major sources of histopathologic processes. Considering that the limited efficacy of these vaccines has been ascribed to local pathogen-induced cytokine dysfunction, we propose to antagonize pathogenic cytokine(s) by high affinity neutralizing auto-Abs triggered by specific anti-cytokine vaccines. As anticipated by theoretical considerations, animal experiments and initial clinical trials showed that anti-cytokine immunization was safe, well tolerated and triggered transient high titers Abs neutralizing pathogenic cytokines but, in contrast to conventional vaccines, no relevant cellular response was observed. Advantages of active versus passive anti-cytokine Ab therapy, particularly for long-term treatments, as those required in AIDS, cancer, allergy and autoimmunity include greater ease of maintaining high Ab titers, lack of anti-antibody responses and low cost.

[Cellular dynamics of pathogenesis induced by bovine leukemia virus]. / Dynamique cellulaire de la pathogenèse induite par le virus de la leucémie bovine.

Bovine leukemia virus (BLV) is the etiological agent of a lymphoproliferative disease in cattle. This retrovirus can also be experimentally transmitted to sheep, in which the pathology is more rapid and more frequent. This review summarizes the current knowledge on the BLV virus and more particularly on its role in lymphocyte homeostasis and induction of pathogenesis. This system has been informative for understanding pathogenesis induced by human T-lymphotropic virus type I (HTLV-1).

Goat lymphosarcoma from bovine leukemia virus.

A goat given inoculations of sheep lymphocytes from cultures that produced bovine leukemia virus (BLV) died 8 years later with lymphosarcoma. The tumors were located in various lymph nodes, the mesentery, omentum, body wall, and retrobulbar tissues. The BLV had been cultured from lymphocytes during the first year after the goat's infection, and persisting BLV antibodies could be demonstrated when the animal was 7.5 years old. BLV provirus was identified by molecular hybridization in the DNA of the goat tumors at the above five locations. The tumors were similar to those found in lymphosarcoma of the adult bovine type (BLV associated). Normal goat liver, normal calf thymus, and calf-type lymphosarcoma (not BLV associated) served as negative controls. Our serologic, histologic, and molecular hybridization studies are evidence that the lymphosarcoma was induced BLV.

Selective activation of cervical microvascular endothelial cells by human papillomavirus 16-e7 oncoprotein.

BACKGROUND: Human papillomavirus type 16 (HPV16) is strongly implicated in the etiology of cervical cancer, with the expression of HPV16-encoded E7 oncoprotein in infected epithelial cells contributing to their malignant transformation. Although nuclear E7 interacts with several nuclear targets, we have previously shown that extracellular E7 can cause suppression of immune cell function. Moreover, cervical microvascular endothelial (CrMVEn) cells treated with E7 increase their expression of adhesion molecules. High levels of some cytokines in serum and in cervicovaginal secretions are associated with the progression of cervical cancer. In this study, we investigated the effects of extracellular E7 on cytokine production and on cytoskeleton structure of CrMVEn cells and vascular endothelial cells from different organs. METHODS: Immunocytochemical staining and flow cytometry techniques were used to detect E7 in endothelial cells incubated with purified E7 protein. Laser scanning confocal microscopy was used to study the E7-induced modification of the endothelial cytoskeleton. An enzyme-linked immunosorbent assay was performed to measure the production of two cytokines, interleukin 6 (IL-6) and interleukin 8 (IL-8), by E7-treated endothelial cells. All statistical tests were two-sided. RESULTS: Extracellular E7 was taken up by CrMVEn cells and localized to the cytoplasm. CrMVEn cells showed a statistically significant (P<.02) increase in the production of IL-6 and IL-8 after treatment with E7 compared with the controls. CrMVEn cells also produced higher levels of these cytokines than did the other endothelial cells (P<.01). E7 also induced marked alterations in the endothelial cytoskeleton of CrMVEn cells as a result of actin fiber polymerization. CONCLUSION: These findings suggest a novel mechanism by which E7, as an extracellular factor, can play a role in the progression and dissemination of cervical cancer via its selective effects on endothelial cells.

Humoral antibody response to bovine leukemia virus infection in cattle and sheep.

In this study, 345 cattle from 7 herds with a history of lymphosarcoma were tested for antibody to BLV antigens by three serological methods, namely immunodiffusion using a bovine leukemia virus glycoprotein with a molecular weight of 60,000 as antigen, and radioimmunoassay using a bovine leukemia virus glycoprotein with a molecular weight of 60,000 and a bovine leukemia virus protein with a molecular weight of 24,000 as antigen. The three tests under comparison agreed for 335 animals, 240 being negative in the three tests, and 95 being positive. Results were variable in ten cases only. Glycoprotein with a molecular weight of 60,000 antibody titers were systematically higher than were protein with a molecular weight of 24,000 antibody titers in bovine sera and milk, as well as in sera of experimentally infected sheep. In the latter case, antibodies to bovine leukemia virus antigens reached maximal values at the animal death in the tumor phase of the disease. Ratios of serum antiglycoprotein titer to milk titer varied between 4 and 117, showing that, if milk pools are to be used in surveys of bovine leukemia virus infection, use of very sensitive techniques of detection is mandatory.

Bovine leukemia virus, a versatile agent with various pathogenic effects in various animal species.

The bovine leukemia virus is the etiological agent of a chronic lymphatic leukemia in cows, sheep, and goats. The same virus seems to induce a kind of wasting disease in experimentally infected rabbits. Antibodies to highly purified bovine leukemia viral Mr 51,000 glycoprotein and Mr 24,000 protein cross-react with human T-lymphotropic virus III/lymphadenopathy-associated virus antigens present in cultured lymphocytes of African patients suffering from acquired immune deficiency syndrome. Bovine leukemia virus has many structural and functional characteristics in common with the human T-lymphotropic viruses. The most striking feature of these retroviruses is the existence of a long open reading frame located at the 3' side of the provirus between the right end of the 3' side of env gene and the left end of the long terminal repeat. It is believed that the long open reading frame protein product acts in trans upon a number of genes to account for the biological effects of the virus.

The amino acid (157-197) peptide segment of bovine leukemia virus p34tax encompass a leucine-rich globally neutral activation domain.

The specific DNA-binding Gal4 amino-terminal portion (amino acids 1 to 147) was fused to protein segments of BLV transactivator p34tax and tested for its capacity to activate CAT-gene expression in mammalian cells. The p34tax peptide segment 157 to 197 encompasses an activating region. The segment is approximately located in the middle of p34tax and is globally neutral (net charge zero). The tax (157-197) domain contains 24% of leucine residues, possibly involved in heterologous protein interactions.

Wild-type p53 down-regulates transcription from oncogenic human papillomavirus promoters through the epithelial specific enhancer.

High-risk Human Papillomavirus (HPV) E6 and E7 immortalizing oncoproteins are expressed from a promoter tightly regulated by an epithelial specific enhancer. To determine if the p53 tumour suppressor protein can modulate the transcription of these genes, we performed co-transfection experiments with plasmids containing the HPV type 16 or 18 long control regions linked to the chloramphenicol acetyl transferase gene, along with p53 expression vectors. Wild-type, but not mutant, murine or human p53 expression vectors reduced the activity of reporter constructs when co-transfected into HeLa or C33 cell lines. Mutations within the HPV TATA boxes did not significantly alter the levels of p53 repression, suggesting a TATA-independent mechanism. Deletion analyses mapped the p53-responsive domain to the constitutive 230 base pair epithelial specific enhancer. In addition, the enhancer could confer p53-mediated repression when placed upstream of a heterologous promoter.

Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.

Sequence analysis of the pig phosphoglucose isomerase gene promoter region.

A cDNA for pig phosphoglucose isomerase (PGI) was used to isolate genomic clones representing the 5' portion of the corresponding gene. A total of 656 bases of the pig PGI gene were obtained that include 5'-flanking information and part of exon 1. A major transcription start was localized at 74 nucleotides from the translation start. The sequence organization of the pig PGI promoter is similar to that of other housekeeping genes. This GC-rich region includes a TATA-like box, two putative Sp1 recognition sites but no CCAAT box.

Orientation into the lipid bilayer of an asymmetric amphipathic helical peptide located at the N-terminus of viral fusion proteins.

The complete amino-acid sequence of viral fusion proteins has been analyzed by the Eisenberg procedure. The region surrounding the cleavage site contains a highly hydrophilic region immediately followed by a membrane-like region. Since the effective cleavage between these two domains seems required to expose the fusogenic domain (located at the N-terminal sequence of the transmembrane like region) which is assumed to interact with the lipid membrane of the host cell, we have focused our analysis on the conformation and mode of insertion of this membrane-like domain in a lipid monolayer. It was inserted as an alpha-helical structure into a dipalmitoylphosphatidylcholine (DPPC) monolayer and its orientation at the lipid/water interface was determined using a theoretical analysis procedure allowing the assembly of membrane components. For each viral protein sequence these N-terminal helical segments oriented obliquely with respect to the lipid/water interface. This rather unusual orientation is envisaged as a prerequisite to membrane destabilization and fusogenic activity.

Are CD4 and Fas peptide identities of gp120 relevant to the molecular basis of AIDS pathogenesis?

The release of virions from HIV-1-infected CD4 cells, although occurring readily as a result of immune activation, does not appear to be the only mechanism mediating T-cell loss in AIDS. Three other interacting HIV-1-induced immune disorders in association with viral release (the source of gp120 molecules) may also account for the constitutive T-cell depletion and functional immune suppression: 1. gp120-induced CD4(+) cell anergy, which can be reproduced in cultures of immune activated normal T-cells in the presence of gp120 or gp120 peptide containing the SLWDQ sequence identity to the CD4 molecule; 2. overproduction of IFNalpha and gamma, 3. activation-driven apoptosis of non infected T-cells. Apoptosis of T-cells could also be: 1. induced by effector components - particularly CTL and lymphotoxins produced by helper T-cells of the anti-Fas autoimmune reaction triggered by gp120 epitopes shared with the Fas/APO-1 molecule; 2. enhanced by IFN overproduction. These molecular mechanisms stress the importance in the progression to AIDS of both the viral load and HIV-induced cytokine dysregulation, including overproduction of IFNalpha, which should be considered as targets in the development of strategies for AIDS prophylaxis and immunotherapy.