Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation

<p>Abstract</p> <p>Background</p> <p>Adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1); however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated.</p> <p>Results</p> <p>We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R) in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1α (MIP-1α), a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was enhanced due to activation of lymphocytes by HTLV-1 infection.</p> <p>Conclusion</p> <p>These data demonstrate that PTHrP and its receptor are up-regulated specifically during immortalization of T-lymphocytes by HTLV-1 infection and may facilitate the transformation process.</p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-3

At weeks 1, 3, 5, 7, 9, 11, and 13 following HTLV-1 infection in the presence or absence of IL-2 compared to day 0; controls 1 and 4 are PBMC-1 and PBMC-2; controls 2 and 5 are PBMC-1 and PBMC-2 stimulated with PHA; controls 3 and 6 are PBMC-1 and PBMC-2 stimulated with IL-2 for one week. ANOVA with Dunnett's tests were used to analyze the data from PTH1R RT-PCR quantification (bar graph shown at the bottom of the panel). The PTH1R levels were significantly greater at weeks 5, 7, 9, and 13 (p < 0.05; indicated by asterisks in the figure). PTH1R expression in HTLV-1-infected T-cells and ATLL cells. Lanes represent: (1) Jurkat (2) MT-2 (3) SLB-1 (4) HUT102 (5) C8166 (6) MET-1 (7) RV-ATL (8) HT-1RV cells. The data showed that PTH1R expression was very low or absent in the ATLL cells (MET-1 and RV-ATL) compared to HTLV-1-infected T-cell lines (MT-2, SLB-1 and HT-1RV). Jurkat T-cells were used as a negative control. βM was used a loading control.<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-4

Malized to human βM gene expression.<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-6

The results showed that MIP-1α expression was up-regulated in PBMCs following stimulation with PHA or IL-2. HTLV-1 infection markedly up-regulated MIP-1α expression in the first week after infection which demonstrated that HTLV-1 infection activated the lymphocytes. MIP-1α levels were significantly different across groups and time. Overall, MIP-1α levels significantly decreased over time (p < 0.0001). After using Dunnett's method to adjust for multiple comparisons, the HTLV-1-treated groups had significantly higher MIP-1α levels than the PBMC group (p < 0.0001).<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-7

(0–13 weeks after co-cultivation) and growth curves are shown. PBMCs were infected with HTLV-1 in the presence of IL-2 (10 U/mL; supplemented from day 1 following HTLV-1 infection) or in the absence of IL-2. PBMCs with no stimulation, PBMCs stimulated with PHA and IL-2 irradiated SLB-1 cells served as controls. The results showed that only HTLV-1-infected cells continued to proliferate beyond 6 weeks in culture. Viable cell numbers were significantly different over time between treatment groups (p < 0.0001). While the PBMC+HTLV-1+IL-2 group cell numbers increased slightly over time, the remaining group cell numbers decreased over time, but the PBMC+HTLV-1 group cell numbers decreased only slightly. After using Dunnett's method to adjust for multiple comparisons, the HTLV-1-treated groups both had significantly higher cell numbers than the PBMC (control) group (p < 0.0001).<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-5

12, p13, p30, HBZ, Rex and Tax. The quantity of the expression plasmid is indicated in μg. Bars represent the mean ± SD of three independent samples. Relative Luc/Gal units were significantly different across groups (p = 0.0002). After adjusting for multiple times of comparison, P2/P3Luc+Rex group and P2/P3Luc+ACH group had significantly greater relative Luc/Gal units than the P2/P3Luc group (p = 0.0006, p = 0.0012, respectively; indicated by asterisks in the figure).<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-2

Ene expression. Specific PTHrP-promoter initiated transcripts are shown for 0, 3, 7 and 13 weeks post co-culture in the presence of IL-2 (A), in the absence of IL-2 (B) and for MT-2 cells (C). The data showed that PTHrP was up-regulated in PBMCs following HTLV-1 infection by the activation of both the P2 and P3 promoters.<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-0

(0–13 weeks after co-cultivation) and growth curves are shown. PBMCs were infected with HTLV-1 in the presence of IL-2 (10 U/mL; supplemented from day 1 following HTLV-1 infection) or in the absence of IL-2. PBMCs with no stimulation, PBMCs stimulated with PHA and IL-2 irradiated SLB-1 cells served as controls. The results showed that only HTLV-1-infected cells continued to proliferate beyond 6 weeks in culture. Viable cell numbers were significantly different over time between treatment groups (p < 0.0001). While the PBMC+HTLV-1+IL-2 group cell numbers increased slightly over time, the remaining group cell numbers decreased over time, but the PBMC+HTLV-1 group cell numbers decreased only slightly. After using Dunnett's method to adjust for multiple comparisons, the HTLV-1-treated groups both had significantly higher cell numbers than the PBMC (control) group (p < 0.0001).<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p