[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.

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.

[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).

Therapeutic vaccine to control stromal tumor-induced immunosuppression in human uterine cervixc cancer.

Cancer cells may escape immune surveillance by secreting in their microenvironment soluble factors that may locally paralyze the stromal effector immune cells. In the human uterine cervix cancer, HPV-16 E7 protein, released in the stroma, should contribute to cancer cells immune escape since this protein inhibits the cellular immune response to recall antigens or alloantigens and strongly enhances the release of immunosuppressive cytokines by APCs. This prompted us to prepare a therapeutic vaccine triggering anti-E7 neutralizing Abs to antagonize the E7-induced stromal immunosuppressive effects and allow cellular immune reaction towards cancer cells including specific CTLs, induced by conventional vaccine, to be effective. Since HPV-16 is a mucosotropic virus, this therapeutic vaccine has been prepared to generate systemic as well as mucosal immunity.

Cervical response to vaccination against HPV16 E7 in case of severe dysplasia.

OBJECTIVE: To evaluate the tolerance to vaccination against human papillomavirus (HPV)16 E7 (in SB adjuvant ASO2B) and its histological and immunohistological effects on HPV16 associated high-grade cervical dysplasias associated with HPV16. STUDY DESIGN: Five patients with histologically demonstrated severe cervical dysplasia (CIN3) HPV16 positive were injected three times before conization was performed 2 months after the first injection. We studied cytological, histological, proliferative pattern and immune profile before and after vaccination. The slides were compared with those obtained from non-injected patients. RESULTS: The injections were well tolerated and the specimens displayed a limited regression of the lesions. Nevertheless, massive CD4 and CD8 T cell lymphocytic infiltration was noticed after vaccination. DISCUSSION: We conclude that the vaccination we used provides an obvious immune histological reaction in the HPV infected cervix and that the 2 months delay before the final step (conization) is done is probably too short.

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.

[Gene therapy]. / La thérapie génique.

Gene therapy is a young scientific discipline, full of promises. Recently a major success was encountered through the correction of the SCID-X1 genetic defect in affected children. Gene therapy is also successfully developing in experimental oncology.

Targeting Tat and IFN(alpha) as a therapeutic AIDS vaccine.

Evolution to AIDS is characterized by a progressive cellular immune suppression. Although there is substantial evidence for several mechanisms involved in disrupting the immune response by induction of apoptosis in responder cells by contact with infected cells, we propose that humoral factors also play a role, and that one such factor is the extracellular form of the human immunodeficiency virus (HIV)-1 Tat protein and another is IFN(alpha). Both Tat and interferon-alpha (IFN(alpha)) inhibit antigen-stimulate T-cell proliferation, and specific anti-Tat and/or anti-IFN(alpha) Abs prevent generation of HIV-1-induced suppressor cells. We propose that high titer anti-Tat and/or anti-IFN(alpha) Abs, neutralizing extracellular Tat, and/or IFN(alpha), induced by vaccines described here, antagonize HIV-1-induced immunosuppression. Innocuous vaccines were prepared by using inactivated but immunogenic Tat (Toxoid) and inactivated and immunogenic IFN(alpha) (kinoid) derivatives. Both Tat Toxoid and IFN(alpha) kinoid were well tolerated and elicited specific neutralizing antibodies (Abs) in mice, monkeys, and seronegative and HIV-1-infected individuals.

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.

Toward a new generation of vaccines: the anti-cytokine therapeutic vaccines.

Pathological conditions, such as cancers, viral infections, and autoimmune diseases, are associated with abnormal cytokine production, and the morbidity associated with many medical disorders is often directly a result of cytokine production. Because of the absence of negative feedback control occurring in some pathophysiologic situations, a given cytokine may flood and accumulate in the extracellular compartment of tissues or tumors thereby impairing the cytokine network homeostasis and contributing to local pathogenesis. To evaluate whether the rise of anti-cytokine Abs by vaccination is an effective way to treat these pathological conditions without being harmful to the organism, we have analyzed each step of the cytokine process (involving cytokine production, target response, and feedback regulation) and have considered them in the local context of effector--target cell microenvironment and in the overall context of the macroenvironment of the immune system of the organism. In pathologic tissues, Abs of high affinity, as raised by anti-cytokine vaccination, should neutralize the pool of cytokines ectopically accumulated in the extracellular compartment, thus counteracting their pathogenic effects. In contrast, the same Abs should not interfere with cytokine processes occurring in normal tissues, because under physiologic conditions cytokine production by effector cells (induced by activation but controlled by negative feedback regulation) does not accumulate in the extracellular compartment. These concepts are consistent with results showing that following animal and human anti-cytokine vaccination, induction of high-affinity Abs has proven to be safe and effective and encourages this approach as a pioneering avenue of therapy.

Human papillomavirus-16-E7 oncoprotein enhances the expression of adhesion molecules in cervical endothelial cells but not in human umbilical vein endothelial cells.

OBJECTIVES: E7 is one of the oncoproteins encoded by human papillomavirus-16 (HPV-16), the major etiologic factor responsible for cervical cancer. Human papillomavirus-16-E7 expressed by human uterine cervix carcinoma cells is also released in the extracellular compartment where it induces immune suppression. We investigated whether E7 was also responsible for the enhanced endothelial adhesiveness required in cancer progression. STUDY DESIGN/METHODS: We treated cervical microvascular endothelial cells (CrMVEn) and human umbilical vein endothelial cells (HUVEC) with E7, tumor necrosis factor-alpha (TNF-alpha), and hydrogen peroxide (H2O2) and measured the expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) by fluorescent-activated cell sorter analysis. RESULTS: E7 strongly induced the expression of E-selectin, ICAM-1, and VCAM-1 in CrMVEn, but not in HUVEC. Tumor necrosis factor-alpha further increased the endothelial expression of adhesion molecules in CrMVEn. Hydrogen peroxide pre-treatment resulted in an enhanced ICAM-1 and a decreased E-selectin and VCAM-1 expression. We also show indirect effects when endothelial cells were stimulated with the supernatant of E7-pretreated macrophages. CONCLUSIONS: These results show that HPV-16-E7 oncoprotein strongly induces adhesion molecules expression in organ-specific endothelial cells.

Suboptimal enhancer sequences are required for efficient bovine leukemia virus propagation in vivo: implications for viral latency.

Repression of viral expression is a major strategy developed by retroviruses to escape from the host immune response. The absence of viral proteins (or derived peptides) at the surface of an infected cell does not permit the establishment of an efficient immune attack. Such a strategy appears to have been adopted by animal oncoviruses such as bovine leukemia virus (BLV) and human T-cell leukemia virus (HTLV). In BLV-infected animals, only a small fraction of the infected lymphocytes (between 1 in 5,000 and 1 in 50,000) express large amounts of viral proteins; the vast majority of the proviruses are repressed at the transcriptional level. Induction of BLV transcription involves the interaction of the virus-encoded Tax protein with the CREB/ATF factors; the resulting complex is able to interact with three 21-bp Tax-responsive elements (TxRE) located in the 5' long terminal repeat (5' LTR). These TxRE contain cyclic AMP-responsive elements (CRE), but, remarkably, the "TGACGTCA" consensus is never strictly conserved in any viral strain (e.g.,AGACGTCA, TGACGGCA, TGACCTCA). To assess the role of these suboptimal CREs, we introduced a perfect consensus sequence within the TxRE and showed by gel retardation assays that the binding efficiency of the CREB/ATF proteins was increased. However, trans-activation of a luciferase-based reporter by Tax was not affected in transient transfection assays. Still, in the absence of Tax, the basal promoter activity of the mutated LTR was increased as much as 20-fold. In contrast, mutation of other regulatory elements within the LTR (the E box, NF-kappa B, and glucocorticoid- or interferon-responsive sites [GRE or IRF]) did not induce a similar alteration of the basal transcription levels. To evaluate the biological relevance of these observations made in vitro, the mutations were introduced into an infectious BLV molecular clone. After injection into sheep, it appeared that all the recombinants were infectious in vivo and did not revert into a wild-type virus. All of them, except one, propagated at wild-type levels, indicating that viral spread was not affected by the mutation. The sole exception was the CRE mutant; proviral loads were drastically reduced in sheep infected with this type of virus. We conclude that a series of sites (NF-kappa B, IRF, GRE, and the E box) are not required for efficient viral spread in the sheep model, although mutation of some of these motifs might induce a minor phenotype during transient transfection assays in vitro. Remarkably, a provirus (pBLV-Delta 21-bp) harboring only two TxRE was infectious and propagated at wild-type levels. And, most importantly, reconstitution of a consensus CRE, within the 21-bp enhancers increases binding of CREB/ATF proteins but abrogates basal repression of LTR-directed transcription in vitro. Suboptimal CREs are, however, essential for efficient viral spread within infected sheep, although these sites are dispensable for infectivity. These results suggest an evolutionary selection of suboptimal CREs that repress viral expression with escape from the host immune response. These observations, which were obtained in an animal model for HTLV-1, are of interest for oncovirus-induced pathogenesis in humans.

Down-modulation of TCR/CD3 surface complexes after HIV-1 infection is associated with differential expression of the viral regulatory genes.

We have investigated the mechanism(s) involved in progressive abrogation of CD3-gamma gene expression after HIV-1 or HIV-2 infection. A comparison of intracellular virus expression with T cell receptor surface density, revealed both high and low levels of viral p24 antigen in the TCR/CD3(hi), TCR/CD3(lo), and TCR/CD3(-) cells. Furthermore, in non-productively infected cells expressing the multiply spliced, virally encoded tat, rev, and nef regulatory gene transcripts, the same progressive loss of surface TCR/CD3 complexes was observed. We treated HIV-1-infected cells with antisense (AS) phosphorothioate oligodeoxynucleotides (P-OdN) targeted to the viral regulatory genes. All of the HIV-1 sequence-specific AS-P-OdN's inhibited intracellular p24 antigen expression in a time- and dose-dependent manner; although, blocking p24 expression alone was not sufficient to modulate TCR/CD3 surface density. Only Tat-AS and Nef-AS were able to delay TCR/CD3 down-modulation on receptor-positive cells or drive receptor up-regulation on receptor-negative cells. In contrast, Rev-AS accelerated TCR/CD3 loss on receptor-positive cells. RT-PCR revealed that Tat-AS and Nef-AS reduce the level of tat, nef, and rev transcripts, while Rev-AS increases the level of tat and nef transcripts in infected cells. Thus, when intracellular conditions favor expression of tat and/or nef in the absence of rev, CD3-gamma gene transcripts and TCR/CD3 surface density are down-modulated.

CD154 costimulated ovine primary B cells, a cell culture system that supports productive infection by bovine leukemia virus.

Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and gamma-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.

[Genes: movers of life and disease]. / Les gènes: moteurs de la vie et de la maladie.

Genes are elements of the genetic material (deoxyribonucleic acid, dna). They code for a protein product. The human genome contains 35,000 genes. Half of our genetic information is inherited from our father, the other half from our mother. Mitrochondria and their DNA are entirely of maternal origin, which allowed Bryan Sykes to establish that modern human subjects can be classified into seven different lineages. The seven daughters of Eve, Bryan Sykes, 2001). Some genes code for structural proteins, some others code for regulatory ones. The efficient control of the cell cycle is carried out by numerous "normal" proteins. Some proteins, mutated on "abnormally" regulated are inducers of diseases, such as cancers or degenerative diseases.

Genetic determinants of bovine leukemia virus pathogenesis.

The understanding of HTLV-induced disease is hampered by the lack of a suitable animal model allowing the study of both viral replication and leukemogenesis in vivo. Although valuable information has been obtained in different species, such as rabbits, mice, rats, and monkeys, none of these systems was able to conciliate topics as different as viral infectivity, propagation within the host, and generation of leukemic cells. An alternate strategy is based on the understanding of diseases induced by viruses closely related to HTLV-1, like bovine leukemia virus (BLV). Both viruses indeed belong to the same subfamily of retroviruses, harbor a similar genomic organization, and infect and transform cells of the hematopoietic system. The main advantage of the BLV system is that it allows direct experimentation in two different species, cattle and sheep.