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Localization, quantification and interaction with host factors of endogenous HTLV-1 HBZ protein in infected cells and ATL.

Raval GU, Bidoia C, Forlani G, Tosi G, Gessain A, Accolla RS - Retrovirology (2015)

Bottom Line: The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein.HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical and Morphological Sciences, School of Medicine, University of Insubria, Via Ottorino Rossi n.9, 21100, Varese, Italy. goutham.uthamraval@uninsubria.it.

ABSTRACT

Background: Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of a severe form of neoplasia designated Adult T cell Leukaemia (ATL). It is widely accepted that the viral transactivator Tax-1 is the major viral product involved in the onset, but not in the maintenance, of neoplastic phenotype, as only 30-40% of ATL cells express Tax-1. It has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, constantly expressed in infected cells and in ATL tumor cells, is also involved in the pathogenesis of leukaemia. The full role played by HBZ in oncogenesis is not clarified in detail also because of the limited availability of tools to assess quantitative expression, subcellular location and interaction of HBZ with host factors in ATL.

Results: By the use of the first reported monoclonal antibody against HBZ, 4D4-F3, generated in our laboratory it has been possible to carefully assess for the first time the above parameters in HTLV-1 chronically infected cells and, most importantly, in fresh leukemic cells from patients. Endogenous HBZ is expressed in speckle-like structures localized in the nucleus. The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein. HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.

Conclusions: The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.

No MeSH data available.


Related in: MedlinePlus

Quantification of endogenous HBZ expression in HTLV-1 chronically infected and in ATL tumor cells. a Three serial dilutions of purified GST-tagged HBZ protein (HBZ.GST) in nanograms (ng) were prepared in lysis buffer, run in SDS-PAGE gel, blotted in nitrocellulose filter, and filter reacted with the 4D4-F3 anti-HBZ mAb. Similarly, 40 μl of 1 ml cell lysates from C5MJ, ATL-2s, PH961 patient ATL tumor cells [PH961(a) and PH96(b)], two distinct cell lysates of equivalent number of cells, 293T cells transfected with HBZ-myc (293T.HBZ) and Jurkat cells, were run in the same gel and processed as above. Cell lysates were from the same cells analyzed by confocal microscopy and shown in Figure 1. It must be underlined that in order to obtain comparable western blots, C5MJ, ATL-2s and PH961 patient cell lysates were prepared from 10 times more cells as compared to 293T.HBZ cell lysates; this corresponded to 330.000 cells for the first three cell lysates vs 33.000 cells for 293T.HBZ cell lysate. Jurkat cell lysate was from 35.000 cells. b The expression of endogenous α-tubulin, used as internal control of sample loading. In this case, lysates from equal number of cells of the various samples were loaded in the gel. c The calculated amounts of HBZ in picograms/cell and in number of molecules/cell for the distinct cell sample are listed.
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Fig4: Quantification of endogenous HBZ expression in HTLV-1 chronically infected and in ATL tumor cells. a Three serial dilutions of purified GST-tagged HBZ protein (HBZ.GST) in nanograms (ng) were prepared in lysis buffer, run in SDS-PAGE gel, blotted in nitrocellulose filter, and filter reacted with the 4D4-F3 anti-HBZ mAb. Similarly, 40 μl of 1 ml cell lysates from C5MJ, ATL-2s, PH961 patient ATL tumor cells [PH961(a) and PH96(b)], two distinct cell lysates of equivalent number of cells, 293T cells transfected with HBZ-myc (293T.HBZ) and Jurkat cells, were run in the same gel and processed as above. Cell lysates were from the same cells analyzed by confocal microscopy and shown in Figure 1. It must be underlined that in order to obtain comparable western blots, C5MJ, ATL-2s and PH961 patient cell lysates were prepared from 10 times more cells as compared to 293T.HBZ cell lysates; this corresponded to 330.000 cells for the first three cell lysates vs 33.000 cells for 293T.HBZ cell lysate. Jurkat cell lysate was from 35.000 cells. b The expression of endogenous α-tubulin, used as internal control of sample loading. In this case, lysates from equal number of cells of the various samples were loaded in the gel. c The calculated amounts of HBZ in picograms/cell and in number of molecules/cell for the distinct cell sample are listed.

Mentions: We further investigated whether the 4D4-F3 antibody could be used to estimate the amount of HBZ protein present in HTLV-1 infected and in ATL tumor cell lines. To this purpose, increasing amounts of purified GST-tagged HBZ protein were used to construct a standard curve by western blotting with the 4D4-F3 antibody; specific bands were then quantified by densitometry. As little as two ng of HBZ protein could be clearly detected by this approach (Figure 4a). Subsequently we performed western blot analysis of cell lysates from a fixed number of 293T cells transfected with the standardized dose of 3 μg of untagged HBZ DNA (Figure 4b) and plot the corresponding densitometry data on the curve constructed with the GST-tagged HBZ. This allowed defining a provisional value of the amount of HBZ present in the transfected cells on a per-cell basis. This value was calculated by taking into account the average percentage of HBZ transfected cells as shown in Figure 3, and it was found to be 20.36 × 10−2 pg/cell, corresponding to 783.000 molecules/cell. Similar experiments were performed with cell lysates of C5MJ and ATL-2s cell lines, as well as PH961 patient cells (Figure 4a). It must be stressed that in order to visualize by western blot specific HBZ bands in C5MJ, ATL-2s and in the PH961 patient cell lysates, comparable for intensity with HBZ-transfected 293T cells, it was necessary to process 10 times more cells. Assuming a molecular weight of 26 kDa for endogenous HBZ, after appropriate comparison by plotting densitometry values on the standard curve it was estimated that HBZ-positive C5MJ or ATL-2s cell lines contained an average of 0.65 × 10−2 pg/cell corresponding to 33.385 molecules/cell, and 1.03 × 10−2 pg/cell corresponding to 39.615 molecules/cell, respectively. For patients PH961 cells it was estimated an average of 0.45 × 10−2 pg/cell corresponding to 17.461 molecules/cell (Figure 4c). Therefore, with all the limitation of the analysis used, we could conclude that the amount of the endogenously made HBZ molecules in freshly isolated ATL cells, in ATL cell line and in a chronically infected cell line was comparatively much lower (20- to 50-fold lower) than the one observed in transiently transfected 293T cells.Figure 4


Localization, quantification and interaction with host factors of endogenous HTLV-1 HBZ protein in infected cells and ATL.

Raval GU, Bidoia C, Forlani G, Tosi G, Gessain A, Accolla RS - Retrovirology (2015)

Quantification of endogenous HBZ expression in HTLV-1 chronically infected and in ATL tumor cells. a Three serial dilutions of purified GST-tagged HBZ protein (HBZ.GST) in nanograms (ng) were prepared in lysis buffer, run in SDS-PAGE gel, blotted in nitrocellulose filter, and filter reacted with the 4D4-F3 anti-HBZ mAb. Similarly, 40 μl of 1 ml cell lysates from C5MJ, ATL-2s, PH961 patient ATL tumor cells [PH961(a) and PH96(b)], two distinct cell lysates of equivalent number of cells, 293T cells transfected with HBZ-myc (293T.HBZ) and Jurkat cells, were run in the same gel and processed as above. Cell lysates were from the same cells analyzed by confocal microscopy and shown in Figure 1. It must be underlined that in order to obtain comparable western blots, C5MJ, ATL-2s and PH961 patient cell lysates were prepared from 10 times more cells as compared to 293T.HBZ cell lysates; this corresponded to 330.000 cells for the first three cell lysates vs 33.000 cells for 293T.HBZ cell lysate. Jurkat cell lysate was from 35.000 cells. b The expression of endogenous α-tubulin, used as internal control of sample loading. In this case, lysates from equal number of cells of the various samples were loaded in the gel. c The calculated amounts of HBZ in picograms/cell and in number of molecules/cell for the distinct cell sample are listed.
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Related In: Results  -  Collection

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Fig4: Quantification of endogenous HBZ expression in HTLV-1 chronically infected and in ATL tumor cells. a Three serial dilutions of purified GST-tagged HBZ protein (HBZ.GST) in nanograms (ng) were prepared in lysis buffer, run in SDS-PAGE gel, blotted in nitrocellulose filter, and filter reacted with the 4D4-F3 anti-HBZ mAb. Similarly, 40 μl of 1 ml cell lysates from C5MJ, ATL-2s, PH961 patient ATL tumor cells [PH961(a) and PH96(b)], two distinct cell lysates of equivalent number of cells, 293T cells transfected with HBZ-myc (293T.HBZ) and Jurkat cells, were run in the same gel and processed as above. Cell lysates were from the same cells analyzed by confocal microscopy and shown in Figure 1. It must be underlined that in order to obtain comparable western blots, C5MJ, ATL-2s and PH961 patient cell lysates were prepared from 10 times more cells as compared to 293T.HBZ cell lysates; this corresponded to 330.000 cells for the first three cell lysates vs 33.000 cells for 293T.HBZ cell lysate. Jurkat cell lysate was from 35.000 cells. b The expression of endogenous α-tubulin, used as internal control of sample loading. In this case, lysates from equal number of cells of the various samples were loaded in the gel. c The calculated amounts of HBZ in picograms/cell and in number of molecules/cell for the distinct cell sample are listed.
Mentions: We further investigated whether the 4D4-F3 antibody could be used to estimate the amount of HBZ protein present in HTLV-1 infected and in ATL tumor cell lines. To this purpose, increasing amounts of purified GST-tagged HBZ protein were used to construct a standard curve by western blotting with the 4D4-F3 antibody; specific bands were then quantified by densitometry. As little as two ng of HBZ protein could be clearly detected by this approach (Figure 4a). Subsequently we performed western blot analysis of cell lysates from a fixed number of 293T cells transfected with the standardized dose of 3 μg of untagged HBZ DNA (Figure 4b) and plot the corresponding densitometry data on the curve constructed with the GST-tagged HBZ. This allowed defining a provisional value of the amount of HBZ present in the transfected cells on a per-cell basis. This value was calculated by taking into account the average percentage of HBZ transfected cells as shown in Figure 3, and it was found to be 20.36 × 10−2 pg/cell, corresponding to 783.000 molecules/cell. Similar experiments were performed with cell lysates of C5MJ and ATL-2s cell lines, as well as PH961 patient cells (Figure 4a). It must be stressed that in order to visualize by western blot specific HBZ bands in C5MJ, ATL-2s and in the PH961 patient cell lysates, comparable for intensity with HBZ-transfected 293T cells, it was necessary to process 10 times more cells. Assuming a molecular weight of 26 kDa for endogenous HBZ, after appropriate comparison by plotting densitometry values on the standard curve it was estimated that HBZ-positive C5MJ or ATL-2s cell lines contained an average of 0.65 × 10−2 pg/cell corresponding to 33.385 molecules/cell, and 1.03 × 10−2 pg/cell corresponding to 39.615 molecules/cell, respectively. For patients PH961 cells it was estimated an average of 0.45 × 10−2 pg/cell corresponding to 17.461 molecules/cell (Figure 4c). Therefore, with all the limitation of the analysis used, we could conclude that the amount of the endogenously made HBZ molecules in freshly isolated ATL cells, in ATL cell line and in a chronically infected cell line was comparatively much lower (20- to 50-fold lower) than the one observed in transiently transfected 293T cells.Figure 4

Bottom Line: The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein.HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical and Morphological Sciences, School of Medicine, University of Insubria, Via Ottorino Rossi n.9, 21100, Varese, Italy. goutham.uthamraval@uninsubria.it.

ABSTRACT

Background: Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of a severe form of neoplasia designated Adult T cell Leukaemia (ATL). It is widely accepted that the viral transactivator Tax-1 is the major viral product involved in the onset, but not in the maintenance, of neoplastic phenotype, as only 30-40% of ATL cells express Tax-1. It has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, constantly expressed in infected cells and in ATL tumor cells, is also involved in the pathogenesis of leukaemia. The full role played by HBZ in oncogenesis is not clarified in detail also because of the limited availability of tools to assess quantitative expression, subcellular location and interaction of HBZ with host factors in ATL.

Results: By the use of the first reported monoclonal antibody against HBZ, 4D4-F3, generated in our laboratory it has been possible to carefully assess for the first time the above parameters in HTLV-1 chronically infected cells and, most importantly, in fresh leukemic cells from patients. Endogenous HBZ is expressed in speckle-like structures localized in the nucleus. The calculated number of endogenous HBZ molecules varies between 17.461 and 39.615 molecules per cell, 20- to 50-fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein. HBZ interacts in vivo with p300 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p300 and JunD but also with CBP and CREB2.

Conclusions: The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role of HBZ during HTLV-1 infection and cellular transformation.

No MeSH data available.


Related in: MedlinePlus