Louis Gazzolo

HTLV-1 HBZ cooperates with JunD to enhance transcription of the human telomerase reverse transcriptase gene (hTERT).

BackgroundActivation of telomerase is a critical and late event in tumor progression. Thus, in patients with adult-T cell leukaemia (ATL), an HTLV-1 (Human T cell Leukaemia virus type 1)-associated disease, leukemic cells display a high telomerase activity, mainly through transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). The HBZ (HTLV-1 bZIP) protein coded by the minus strand of HTLV-1 genome and expressed in ATL cells has been shown to increase the transcriptional activity of JunD, an AP-1 protein. The presence of several AP-1 binding sites in the hTERT promoter led us to investigate whether HBZ regulates hTERT gene transcription.ResultsHere, we demonstrate using co-transfection assays that HBZ in association with JunD activates the hTERT promoter. Interestingly, the -378/+1 proximal region, which does not contain any AP-1 site was found to be responsible for this activation. Furthermore, an increase of hTERT transcripts was observed in cells co-expressing HBZ and JunD. Chromatin immunoprecipitation (ChIP) assays revealed that HBZ, and JunD coexist in the same DNA-protein complex at the proximal region of hTERT promoter. Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter.ConclusionThese observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.

Target cells infected by avian erythroblastosis virus differentiate and become transformed.

Transformation in vitro of bone marrow cells by avian erythroblastosis virus (AEV) gives rise to rapidly growing cells of erythroid nature. Target cells of neoplastic transformation by AEV are recruited among the early progenitors of the erythroid lineage, the burst-forming units-erythroid (BFU-E). They express a brain-related antigen at a high level and an immature antigen at a low level. We show that AEV-transformed cells express low levels of the brain antigen and high levels of the immature antigen. Their response to specific factors regulating the erythroid differentiation indicates that they are very sensitive to erythropoietin. Furthermore, cells transformed by a temperature-sensitive mutant of AEV differentiate into hemoglobin-synthesizing cells 4 days after being shifted to the nonpermissive temperature. All these properties are similar to those of late progenitors of the erythroid lineage, the colony-forming units-erythroid (CFU-E). These results indicate that the AEV-transformed cells are blocked in their differentiation at the CFU-E stage.

Characterization of the hemopoietic target cells for the avian leukemia virus E26

The dual leukemogenic response, involving both the erythroid and myeloid hemopoietic systems in chickens infected with E26 virus, has previously been described (C. Moscovici, J. Samarut, L. Gazzolo, and M. G. Moscovici, 1981. Virology 113, 765-768; K. Radke, H. Beug, S. Kornfeld, and T. Graf, 1982. Cell 31, 643-653). Similarly, the in vitro response of the two lineages resulted in the concomitant transformation and proliferation of erythroblast and myeloblast leukemic cells. The present study, using embryonic tissues at very early stages of development, was valuable in implying that E26 target cells are recruited among uncommitted erythroid-myeloid stem cells as well as myeloid- or erythroid-committed progenitor cells. Therefore, E26 may be the first avian retrovirus capable of interacting with uncommitted hemopoietic precursor cells.

Myeloid and erythroid neoplastic responses to avian defective leukemia viruses in chickens and in quail

Abstract A comparative study of the oncogenic potential of two avian defective leukemia viruses (DLVs) shows that avian erythroblastosis virus induces myeloid leukemia in quail and confirms that E26 causes erythroid leukemia in chickens. These results indicate that the response of birds to these two DLVs depends on the host species and suggest that classification of DLVs based only on in vitro studies may not be definitive.

Early precursors in the erythroid lineage are the specific target cells of avian erythroblastosis virus in vitro

In chickens the erythroid differentiation proceeds from stem cells to erythrocytes through several intermediate steps which have been identified in vivo and in vitro. To determine whether Avian erythroblastosis virus (AEV) is able to transform in vitro either one or several types of these precursors, bone marrow cells were separated by physical and immunological methods. It was found that the target cells which could be transformed in vitro by AEV were cells of light density (1.060-1.065 g/cm3), having a modal sedimentation velocity at unit gravity between 4.0 and 6.0 mm/hr, expressing an immature antigen at a low level and a brain-related antigen at a high level. These results indicated that the target cells of neoplastic transformation by AEV were early erythroid precursors, since these precursors shared the same physical and immunological properties with AEV target cells.

Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope.

The CD4 molecule is known to be the preferential receptor for the HIV‐1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV‐1, we investigated the possible involvement of the interactions between HIV‐1 gp 120 or HIV‐1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti‐CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s‐receptor complexes enters Langerhans cells by a process identified as a receptor‐mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV‐1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1‐day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.

Malondialdehyde production from spermine by homogenates of normal and transformed cells

The oxidation of spermine in vitro by a mixture of polyamine oxidase and diamine oxidase from pig kidney gives rise to malondialdehyde via 3-aminopropanol as the intermediate. Conversely, with spermidine, under similar experimental conditions, no evidence could be obtained for malondialdehyde formation within the limits of sensitivity of the assay (2.0 nmol). The activities of both these enzymes show about a 2-fold increase in normal rat kidney cells (LA31 NRK) transformed by the temperature sensitive mutant of Rous sarcoma virus (LA31) and incubated at the non permissive temperature (39 degrees C) compared to the activities in LA31 NRK at the permissive temperature (33 degrees C). These same enzymatic activities show no temperature dependent changes in normal rat kidney cells (NRK) or in these same cells infected by the wild type virus (NRK B77). In extracts derived from Friend erythroleukemic cells induced to differentiate by dimethyl sulfoxide or hexamethylene bis acetamide, spermine oxidation takes place more efficiently than in non induced cells. A rise in diamine oxidase activity is seen in LA31 NRK (39 degrees C) 12 h after the temperature shift, whereas morphological manifestations of normalcy are seen only at 48 h. The Km of diamine oxidase is 10(-6) M for putrescine and 10(-3) M for 3-aminopropanol. A possible mechanism involving the well documented acetylation of putrescine [23,26] is proposed for diverting intracellular putrescine away from cytosolic diamine oxidase and towards intramitochondrial monoamine oxidase.

Transforming ability of avian defective leukemia viruses in early embryogenesis

The response to infection of chicken hemopoietic cells derived from the early stages of embryogenesis by avian myeloblastosis virus (AMV) and avian erythroblastosis virus (AEV) was investigated. It was found that erythroid progenitor cells were present in the blastoderm at a higher frequency than that of myeloid progenitor cells. These results correlate with the observation that target cells for AEV were found to be more numerous than those for AMV. Therefore, blastoderm cells are of potential value in understanding the mechanisms of oncogenesis at the level of the target cells.

A study of the epithelioid transformation of MC29-infected chicken embryo cells

Abstract Myelocytomatosis virus, strain MC29, exhibits a morphological pattern of transformation in infected chicken embryo cultures (CEC) that is unique among the avian retroviruses. When secondary CEC were infected with this virus, round cell foci of transformed cells were observed together with distinct epithelioid cell foci. The epithelioid transformation in secondary CEC was dependent on the presence of dimethylsulfoxide (DMSO) in the overlay medium. However, when later transfers of CEC were infected, epithelioid transformation was not observed even in the presence of DMSO. The study corroborates the previous findings from other laboratories and our own that the phenotypic expression of transformation observed with MC29 is dependent on the presence of more than one target cell.

Identification and characterization of auxiliary proteins encoded by the STLV-3 retrovirus pX region

The PTLV-3 group includes simian viruses (STLV-3) and the recently identified human viruses (HTLV-3). These viruses display a high percentage (>95%) of sequence identity. Recent studies have shown that the auxiliary proteins of complex retroviruses such as HIV and HTLV-1 are playing an important role in the viral life cycle in vivo. However, it has not been determined yet whether the genome of the Primate T-cell Lymphotropic viruses, type 3 encodes such proteins. n nTo uncover the potential presence of auxiliary proteins, we first extracted RNA from cells either infected with STLV-3 or transfected with a STLV-3 infectious molecular clone. RT-PCR experiments using primers specific of the pX region allowed the amplification of two different doubly spliced mRNAs, one encoding a putative 63 amino-acid protein and another one encoding a putative 79 amino-acid protein. Based on the molecular weight prediction, we named these proteins p8 and p9, respectively. The p8 sequence is present in 90% of all HTLV-3 and STLV-3 strains. The N-Ter 21 amino acid sequence is shared with the corresponding Rex3 sequence. This sequence was found to be homologous to that of Rex1, which contains the nucleolar localization signal, and the RNA binding domain. Interestingly, after transfection of a p8 expression vector, we observed that the protein localized within the nucleolus. We are proceeding to the characterization of p8 subdomains as well as to the functional analyses of the p8 functions. These experiments will allow us to determine whether p8 represent the counterpart of an HTLV-1 auxiliary protein.