Lucas Willems

Valproate, in combination with pemetrexed and cisplatin, provides additional efficacy to the treatment of malignant mesothelioma.

Purpose: Present chemotherapeutic regimens are marginally efficient in tumor cells being particularly resistant to radiotherapy and/or chemotherapy. We hypothesized that unresponsiveness of tumors to conventional therapeutic agents might be due to inappropriate gene expression resulting from epigenetic modifications and leading to transcriptional silencing. The goal of this study was to evaluate the anticancer effect of a histone deacetylase inhibitor, valproate, on mesothelioma cells in combination with pemetrexed and cisplatin, the usual first-line regimen of chemotherapy for this tumor. Experimental Design and Results: We show that valproate augments apoptosis induced by pemetrexed and cisplatin in mesothelioma cell lines and in tumor cells from patients biopsies. Onset of apoptosis involves both extrinsic and intrinsic pathways requiring enzymatic activities of caspases 8 and 9, respectively. Valproate but not suberoylanilide hydroxamic acid efficiently stimulates the production of reactive oxygen species. The free radical scavenger N-acetylcysteine inhibits apoptosis, indicating that reactive oxygen species are major mediators of valproate activity. As expected, valproate alone or combined with pemetrexed and cisplatin triggers hyperacetylation of histone H3. Bid protein processing in truncated t-Bid and cytochrome c release from mitochondria are significantly increased in the presence of valproate, providing a mechanistic rationale for improvement of the proapoptotic efficacy of cisplatin and pemetrexed. Finally, valproate when combined with pemetrexed and cisplatin prevents tumor growth in mouse models of epithelioid mesothelioma. Conclusions: These observations support the potential additional efficacy of valproate in combination with pemetrexed and cisplatin for treatment of malignant mesothelioma.

Cooperation between bovine leukaemia virus transactivator protein and Ha-ras oncogene product in cellular transformation

Human T‐lymphotropic viruses (HTLV‐I and ‐II) and bovine leukaemia virus (BLV) express transactivator proteins able to increase long terminal repeat (LTR) directed viral expression. These transacting factors are though to be involved in the induction of leukaemia by these viruses. Transfection of BLV transactivator p34tax together with Ha‐ras immortalizes and transforms rat embryo fibroblasts, in vitro. The transformed cell induce tumours in nude mice. These data emphasize the causal role exerted by p34tax in in vivo tumorigenesis.

The bovine leukemia virus p34 is a transactivator protein

Recombinant Moloney murine retroviruses containing the BLV post‐envelope long open reading frame were constructed and transfected into the psi 2 packaging cell line. They were shown to encode and to express a 34‐kd protein able to transactivate the BLV long terminal repeat‐directed gene expression in the respective transfected cells. These data demonstrate that the BLV X‐LOR gene encodes a p34 transactivator product. Furthermore, the different cell lines produced infectious recombinant retroviruses capable of transferring X‐LOR genes into recipient cells. The availability of the BLV transactivator protein should allow us to understand the role of the transactivator protein in BLV‐induced leukemogenesis.

Oncoviral bovine leukemia virus G4 and human T-cell leukemia virus type 1 p13(II) accessory proteins interact with farnesyl pyrophosphate synthetase.

ABSTRACT G4 and p13II are accessory proteins encoded by the X region of bovine leukemia virus and human T-cell leukemia virus type 1 (HTLV-1), respectively. Disruption of the G4 and p13II open reading frames interferes with viral spread in animal model systems, indicating that the corresponding proteins play a key role in viral replication. In addition, G4 is oncogenic in primary cell cultures and is absolutely required for efficient onset of leukemogenesis in sheep. To gain insight into the function of these proteins, we utilized the yeast two-hybrid system to identify protein partners of G4. Results revealed that G4 interacts with farnesyl pyrophosphate synthetase (FPPS), a protein involved in the mevalonate/squalene pathway and in synthesis of FPP, a substrate required for prenylation of Ras. The specificity of the interaction was verified by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation experiments. Furthermore, confocal microscopy showed that the subcellular localization of G4 was profoundly affected by FPPS. The G4 protein itself was not prenylated, at least in rabbit reticulocyte lysate-based assays. The domain of G4 required for binding to FPPS was restricted to an amphipathic α-helix rich in arginine residues. Subtle mutation of this α-helix abrogated G4 oncogenic potential in vitro, providing a biological relevance for FPPS-G4 complex formation in cells. Finally, HTLV-1 p13II was also found to specifically interact with FPPS (in yeast as well as in GST pull-down assays) and to colocalize with G4 in mitochondria, suggesting a functional analogy between these oncoviral accessory proteins. Identification of FPPS as a molecular partner for p13II and G4 accessory proteins opens new prospects for treatment of retrovirus-induced leukemia.

Suboptimal Enhancer Sequences Are Required for Efficient Bovine Leukemia Virus Propagation In Vivo: Implications for Viral Latency

ABSTRACT 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-κB, 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-κB, 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-Δ21-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.

Reduction of B cell turnover in chronic lymphocytic leukaemia.

Whether chronic lymphocytic leukaemia (CLL) is a latent or a proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukaemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements indicate that B lymphocytes do divide at significant rates in CLL. However, an important and still unanswered question is whether CLL cells proliferate faster or slower compared with their normal counterparts. This report addressed directly this point and compared B‐cell kinetics in CLL subjects and healthy controls, using a pulse‐chase approach based on incorporation of deuterium from 6,6‐2H2‐glucose into DNA. We confirmed that B cells proliferated at significant levels in CLL but found that the proliferation rates were reduced compared with healthy subjects (mean 0·47 vs. 1·31%/d respectively, P = 0·007), equivalent to an extended doubling time of circulating B cells (147 d vs. 53 d). In conclusion, CLL B cells proliferate at reduced levels compared with healthy controls. CLL is thus characterized by an aberrant B‐cell kinetics with a decrease in cell turnover, an observation that may impact on elaboration of efficient therapeutic strategies.

Investigation of the Susceptibility of Human Cell Lines to Bovine Herpesvirus 4 Infection: Demonstration that Human Cells Can Support a Nonpermissive Persistent Infection Which Protects Them against Tumor Necrosis Factor Alpha-Induced Apoptosis

ABSTRACT Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we performed in vitro experiments to assess the risk and the consequences of human infection by BoHV-4. First, by using a recombinant BoHV-4 strain expressing enhanced green fluorescent protein under the control of the human cytomegalovirus immediate-early gene promoter, we tested 21 human cell lines for their sensitivity and their permissiveness to BoHV-4 infection. These experiments revealed that human cell lines from lymphoid and myeloid origins were resistant to infection, whereas epithelial cells, carcinoma cells, or adenocarcinoma cells isolated from various organs were sensitive but poorly permissive to BoHV-4 infection. Second, by using the HeLa cell line as a model of human cells sensitive but not permissive to BoHV-4 infection, we investigated the resistance of infected cells to apoptosis and the persistence of the infection through cellular divisions. The results obtained can be summarized as follows. (i) BoHV-4 nonpermissive infection of HeLa cells protects them against tumor necrosis factor alpha-induced apoptosis. (ii) BoHV-4 infection of HeLa cells persists in cell culture; however, the percentage of infected cells decreases with time due to erratic transmission of the viral genome through cell division. (iii) BoHV-4 infection has no effect on the rate of HeLa cell division. Altogether, these data suggest that BoHV-4 could infect humans. This study also stresses the importance of considering the insidious effects of nonpermissive infection when the biosafety of animal gammaherpesviruses for humans is being considered.

Overlapping CRE and E Box Motifs in the Enhancer Sequences of the Bovine Leukemia Virus 5′ Long Terminal Repeat Are Critical for Basal and Acetylation-Dependent Transcriptional Activity of the Viral Promoter: Implications for Viral Latency

ABSTRACT Bovine leukemia virus (BLV) infection is characterized by viral latency in a large proportion of cells containing an integrated provirus. In this study, we postulated that mechanisms directing the recruitment of deacetylases to the BLV 5′ long terminal repeat (LTR) could explain the transcriptional repression of viral expression in vivo. Accordingly, we showed that BLV promoter activity was induced by several deacetylase inhibitors (such as trichostatin A [TSA]) in the context of episomal LTR constructs and in the context of an integrated BLV provirus. Moreover, treatment of BLV-infected cells with TSA increased H4 acetylation at the viral promoter, showing a close correlation between the level of histone acetylation and transcriptional activation of the BLV LTR. Among the known cis-regulatory DNA elements located in the 5′ LTR, three E box motifs overlapping cyclic AMP responsive elements (CREs) in U3 were shown to be involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutations of these three E box motifs markedly reduced the inducibility of the BLV promoter by TSA. E boxes are susceptible to recognition by transcriptional repressors such as Max-Mad-mSin3 complexes that repress transcription by recruiting deacetylases. However, our in vitro binding studies failed to reveal the presence of Mad-Max proteins in the BLV LTR E box-specific complexes. Remarkably, TSA increased the occupancy of the CREs by CREB/ATF. Therefore, we postulated that the E box-specific complexes exerted their negative cooperative effect on BLV transcription by steric hindrance with the activators CREB/ATF and/or their transcriptional coactivators possessing acetyltransferase activities. Our results thus suggest that the overlapping CRE and E box elements in the BLV LTR were selected during evolution as a novel strategy for BLV to allow better silencing of viral transcription and to escape from the host immune response.

Bovine Leukemia Virus SU Protein Interacts with Zinc, and Mutations within Two Interacting Regions Differently Affect Viral Fusion and Infectivity In Vivo

ABSTRACT Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor.

Discordance between Bovine Leukemia Virus Tax Immortalization In Vitro and Oncogenicity In Vivo

ABSTRACT Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.