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Redefinition of HBZ localization in ATL
unspliced form of HBZ mRNA,25 corroborated also in HAM/TSP patients and in AC that have exclusive HBZ protein cytoplasmic localization.26,27 Conversely, the ATL- 2 leukemic cell line and leukemic cells of a previously described patient (PH1505), displaying a predominant HBZ nuclear localization, showed a more abundant or similar unspliced versus spliced HBZ, suggesting a correla- tion between subcellular protein localization and distinct forms of alternatively spliced HBZ mRNA.
Taken together, these results show for the first time that the dual cytoplasmic/nuclear localization of HBZ is an exclusive feature of ATL, giving further credit to the hypothesis of subcellular re-localization of HBZ in leuke- mogenesis (Figure 6).
The high percentage of HBZ-positive cells observed in chronic ATL, a situation usually characterized by a low number of leukemic cells,47 as well as the exclusive cyto- plasmic localization of HBZ found in two chronic ATL patients, remain to be clarified. Are the described condi- tions the mirror of a specific step of the neoplastic process such as the anticipation of an acute phase of the disease or a phase in which HBZ is progressively relocated into the nucleus without any modification of the clinical status, or, provocatively, the manifestation of novel disease sub- groups? The first hypothesis seems unlikely as clinical fol- low-up suggests that these patients have not evolved toward an aggressive phenotype, indicating that other fac- tors, possibly involved in the cytoplasmic dislocation of HBZ, are needed to mark the transition versus a different clinical state of ATL.
Previous studies of our group have clearly demonstrated that both in asymptomatic carriers and HAM/TSP patients
the exclusive HBZ cytoplasmic localization was due to a retention in this subcellular compartment, as inhibition of CRM-1 dependent nuclear export by LMB did not result in nuclear retention of the viral protein.26,27 It was therefore important to assess whether in cells of ATL patients show- ing both cytoplasmic and nuclear localization, HBZ could freely shuttle between the two subcellular compartments. This was neither the case in acute nor chronic ATL, sug- gesting, besides the above described association of spliced-cytoplasmic versus unspliced-nucleus, the exis- tence of an active mechanism of cytoplasmic HBZ reten- tion even in presence of a quota of nuclear protein, and irrespective of the clinical form of the disease (Figure 6). One possible candidate for the cytoplasmic retention of HBZ could be calreticulin previously shown to regulate Tax-1 nuclear export.40 However, at least for the patients analyzed, HBZ was not clearly confined in a calreticulin subcellular compartment, while Tax-1 partially colocal- ized with calreticulin. A more in-depth analysis with an increased number of acute and chronic ATL subjects might further clarify this aspect.
Recent results have suggested that HBZ can be retained in the cytoplasm by interacting with the T cell- specific molecule THEMIS.48 Conversely, we showed that this did not explain the cytoplasmic retention of HBZ in cells of HAM/TSP patients.26 Thus, although in ATL a partial involvement of THEMIS in the retention of HBZ in the cytoplasm cannot be excluded, it is certainly important to further investigate the possibility that dif- ferent cytoplasmic anchoring molecules or molecular complexes may also be involved in the cytoplasmic local- ization of HBZ in ATL. Whatever the mechanism, it is
Figure 6. Subcellular localization of HTLV-1 HBZ protein during adult T-cell leukemia-lymphoma progression. Primary infection of T cells by HTLV-1 is characterized by the expression and localization of HBZ protein exclusively in the cytoplasm, at variance with Tax-1 that can be found both in the cytoplasm and nucleus. This fea- ture is conserved during the progression to chronic neurologic inflammatory HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) disease possibly due to retention by a cytoplasmic factor present in both asymptomatic carriers and HAM/TSP patients. Leukemogenesis, instead, is marked by the progressive translocation of HBZ from the cytoplasm to the nucleus, both in chronic and in acute adult T-cell leukemia-lymphoma (ATL). On the basis of our results presented here, it is likely that HBZ cytoplasmic localization in ATL is mediated by the same retention factor/mechanism present in asymptomatic carriers and HAM/TSP, that is gradually lost during neoplastic transformation allowing HBZ protein to dislocate into the nucleus.
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