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Redefinition of HBZ localization in ATL
of the disease, prognosis of ATL remains poor.9,10 It is gen- erally accepted that the oncogenic process leading to overt ATL proceeds through a multistep mode in which addi- tional mutations are accumulated in the neoplastic clones.6,11,12 However, due to the specificity of ATL pheno- type in comparison to other T- cell neoplasias, it is likely that virus infection and viral protein expression play a role in oncogenesis.13
Two viral proteins, the viral transactivator Tax-1 and the HTLV-1 basic-zipper factor (HBZ), have shown oncogenic activities in vitro and in vivo in experimental animal models.14,15 Tax-1 seems to be crucial in the onset of the oncogenic process mostly by disarranging several cellular activation pathways and particularly the NF-kB path- way.16-18 However, Tax-1 expression is lost in most of ATL cases suggesting that it might be dispensable for maintain- ing the neoplastic state. Loss of Tax-1 expression can be generated by both genetic lesions and epigenetic mecha- nisms.19,20
Onset of ATL clones with defects in Tax-1 expression are probably favored by immunological selection. Indeed, Tax-1 is strongly immunogenic and a preferential target of cytotoxic T cells (CTL).21,22 In contrast to Tax-1, the nega- tive strand-encoded HBZ,23 is expressed at all stages of infection and neoplastic transformation,24 suggesting that it may be required for the maintenance of the oncogenic process. Interestingly, it has been shown that not only HBZ protein but also HBZ mRNA may be involved in the HTLV-1-mediated oncogenesis.25 Nevertheless, the inti- mate mechanism by which HBZ participates to the HTLV- 1-mediated neoplastic transformation still remains elusive. The subcellular distribution of HBZ in the various phases of the disease may be relevant as recent studies of our group demonstrated a peculiar and exclusive cytoplasmic distribution of HBZ protein in peripheral blood mononu- clear cells (PBMC) of both asymptomatic carriers (AC)26 and HAM/TSP patients27,28 compared to a preferential nuclear localization in a small sample of ATL cells.29
These findings were unprecedented and rather unex- pected as previous studies, mostly performed in HBZ transfected cells, showed an exclusively nuclear distribu- tion of the protein.30,31
Our studies, thus suggested that neoplastic transforma- tion of HTLV-1-infected cells may be accompanied by a unidirectional displacement of HBZ from the cytoplasm to the nucleus.
Thus, we analyzed HBZ localization in fresh leukemic cells of ATL patients, representative of distinct clinical forms of the disease, to investigate potential HBZ transi- tion from the cytoplasm to the nucleus and, in case of transition, to correlate it with distinct ATL clinical entities.
Here we show that leukemic cells from eight ATL patients can express HBZ not only in the nucleus but also in the cytoplasm. Neoplastic transformation is, thus accompanied by a dichotomy of HBZ localization and the exclusively cytoplasmic localization, as observed in AC and in HAM/TSP patients, is progressively modified to include nuclear localization of the protein.
Methods
Cells and ethics statement
This study was approved by the Ethical Committee (CPP Ile de FranceII, CNIL: number 1692254, registration number 000001072)
and all surviving patients gave written informed consent. The study had a retrospective observational design. Patients with acute or chronic ATL at diagnosis were analyzed. See the Online Supplementary Appendix for further details.
HTLV-1 proviral load measurement and determination of unspliced versus spliced HBZ mRNA
Specific information on proviral load measurement and detec- tion of unspliced versus spliced HBZ RNA is provided in the Online Supplementary Appendix.
Cell treatments
See the Online Supplementary Appendix for details.
Immunofluorescence, flow cytometry and confocal microscopy
Frozen vials containing PBMC were thawed by immediate pas- sage from liquid nitrogen to a water bath set at 37°C. Cells were washed with warm RPMI medium and immediately processed for immunofluorescence and flow cytometry analysis or for confocal microscopy as previsouly described.26,32,33 Additional information on antibodies used for detection of specific markers is reported in the Online Supplementary Appendix.
Results
Cell surface phenotype and HBZ subcellular localization in acute adult T-cell leukemia-lymphoma patients: HBZ can reside in the cytoplasm
We first investigated a group of four clinically defined acute ATL patients (namely PH131213, PH140126, PH160822 and PH1612N07).
Preliminary cell surface phenotype of PBMC from these patients showed that CD4+ T cells were the predominant, if not the total, cell subpopulation present in peripheral blood (Online Supplementary Figure S1). Interestingly, expression of the CD4 marker varied in the analyzed ATL patients, with strong (PH140126), moderate (PH131213 and PH1612N07) and low (PH160822) expression. This, however, did not correlate with expression of the CD3 marker which was either very low or absent in PBMC of these patients, a result reminiscent of previous findings reported by our group and others.27,29,34,35 The T-cell activa- tion marker CD25 was expressed in two (PH131213 and PH140126) of four patients’ ATL cells, however, without correlation with the other T-cell activation marker, HLA class II. Expression and subcellular localization of HBZ was then analyzed by immunofluorescence and confocal microscopy Surprisingly, in contrast to what has been reported to date, in all four patients HBZ was predomi- nantly found in the cytoplasm where it appeared as large dots (PH1612N07 and PH160822) with the tendency to converge in diffuse areas (PH131213 and PH140126) around the nucleus (Figure 1A). Interestingly, in PH1612N07 HBZ-positive cells showed the typical flower- like phenotype described in acute ATL. HBZ cytoplasmic localization was confirmed by co-staining with the cyto- plasmic marker vimentin and the nuclear marker DRAQ5 (Figure 1B).
Of note, a wide variation in the percentage of HBZ pos- itive cells was found in the four acute ATL patients, rang- ing from as low as 8.5% (PH131213) to as high as 83% (PH140126) of PBMC leukemic cells (Online Supplementary Table S1). When present, HBZ nuclear localization was
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