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Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways.

Agbottah E, Yeh WI, Berro R, Klase Z, Pedati C, Kehn-Hall K, Wu W, Kashanchi F - AIDS Res Ther (2008)

Bottom Line: The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us.The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage.The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target both NF-kappaB and CDK complex and the G1/S border, might be promising new agents in the treatment of these infected patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, George Washington University School of Medicine, Washington, District of Columbia 20037, USA.

ABSTRACT
Human T-cell leukemia virus type-1 (HTLV-1) induces adult T-cell leukemia/lymphoma (ATL/L), a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-kappaB and the cell cycle pathways. The observation that NF-kappaB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-kappaB and CDK inhibitors (total of 35 compounds) to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKbeta kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration) value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target both NF-kappaB and CDK complex and the G1/S border, might be promising new agents in the treatment of these infected patients.

No MeSH data available.


Related in: MedlinePlus

BMS-345541 inhibition of IKKβ in HTLV-1 infected cell. A) BMS-345541 reduced IKKβ activity in C8166 cells. Equal amount (1 mg) of cytoplasmic proteins was immunoprecipitated with anti-IKKβ antibody and mixed with 1 μM BMS-345541. The IKKβ activities were examined by in vitro kinase assay using GST-IκBα as a substrate. The [γ-32P]-labeled IκB-α protein was visualized by autoradiography. The IKKβ activities were quantitated by ImageQuant software. The bottom panel shows a commassie blue staining of GST-IκBα to show equal amount of substrate in each reaction. B) BMS-345541 inhibited IKKβ activity in C8166 cells in dose-dependent manner; however, Purvalanol A had no effect on IKKβ. Kinase assay were performed as described above using 0.01, 0.1, and 1 μM of BMS-345541 and 1, 10 μM of Purvalanol A. The stained gel below is a representative of the kinase reaction.
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Figure 1: BMS-345541 inhibition of IKKβ in HTLV-1 infected cell. A) BMS-345541 reduced IKKβ activity in C8166 cells. Equal amount (1 mg) of cytoplasmic proteins was immunoprecipitated with anti-IKKβ antibody and mixed with 1 μM BMS-345541. The IKKβ activities were examined by in vitro kinase assay using GST-IκBα as a substrate. The [γ-32P]-labeled IκB-α protein was visualized by autoradiography. The IKKβ activities were quantitated by ImageQuant software. The bottom panel shows a commassie blue staining of GST-IκBα to show equal amount of substrate in each reaction. B) BMS-345541 inhibited IKKβ activity in C8166 cells in dose-dependent manner; however, Purvalanol A had no effect on IKKβ. Kinase assay were performed as described above using 0.01, 0.1, and 1 μM of BMS-345541 and 1, 10 μM of Purvalanol A. The stained gel below is a representative of the kinase reaction.

Mentions: We next focused our attention on BMS-345541 and asked whether this drug could inhibit the IKKβ kinase activity on its substrate IκBα. We immunoprecipitated (IP) IKKβ from both CEM (uninfected) and C8166 (infected) cells and used them in an in vitro kinase assays in the presence or absence of BMS-345541 (1.0 μM). Results are shown in Figure 1A where C8166 cells had far stronger IKKβ kinase activity as compared to CEM cells (compare lanes 2 to 4). Active kinases that were incubated with BMS-345541 showed a reduction of activity from both infected and uninfected cell extracts. However, the inhibition was much more dramatic with kinases isolated from HTLV-1 infected cells. We next titrated various levels of BMS-345541 for both kinases in our in vitro assay. Results are shown in Panel B where 0.01, 0.1, and 1.0 μM of BMS-345541 were used for a complete range of titrations. Interestingly, at 0.1 μM there was a significant reduction in the kinase activity from infected cells (lane 4 compared to lane 9). A control drug, Purvalanol A, which is a CDK inhibitor, did not inhibit the IKKβ kinase activity obtained from infected cells. Collectively, these results indicate that IKKβ from infected cells is much more sensitive to BMS-345541 as compared to IKKβ from uninfected cells.


Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways.

Agbottah E, Yeh WI, Berro R, Klase Z, Pedati C, Kehn-Hall K, Wu W, Kashanchi F - AIDS Res Ther (2008)

BMS-345541 inhibition of IKKβ in HTLV-1 infected cell. A) BMS-345541 reduced IKKβ activity in C8166 cells. Equal amount (1 mg) of cytoplasmic proteins was immunoprecipitated with anti-IKKβ antibody and mixed with 1 μM BMS-345541. The IKKβ activities were examined by in vitro kinase assay using GST-IκBα as a substrate. The [γ-32P]-labeled IκB-α protein was visualized by autoradiography. The IKKβ activities were quantitated by ImageQuant software. The bottom panel shows a commassie blue staining of GST-IκBα to show equal amount of substrate in each reaction. B) BMS-345541 inhibited IKKβ activity in C8166 cells in dose-dependent manner; however, Purvalanol A had no effect on IKKβ. Kinase assay were performed as described above using 0.01, 0.1, and 1 μM of BMS-345541 and 1, 10 μM of Purvalanol A. The stained gel below is a representative of the kinase reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483717&req=5

Figure 1: BMS-345541 inhibition of IKKβ in HTLV-1 infected cell. A) BMS-345541 reduced IKKβ activity in C8166 cells. Equal amount (1 mg) of cytoplasmic proteins was immunoprecipitated with anti-IKKβ antibody and mixed with 1 μM BMS-345541. The IKKβ activities were examined by in vitro kinase assay using GST-IκBα as a substrate. The [γ-32P]-labeled IκB-α protein was visualized by autoradiography. The IKKβ activities were quantitated by ImageQuant software. The bottom panel shows a commassie blue staining of GST-IκBα to show equal amount of substrate in each reaction. B) BMS-345541 inhibited IKKβ activity in C8166 cells in dose-dependent manner; however, Purvalanol A had no effect on IKKβ. Kinase assay were performed as described above using 0.01, 0.1, and 1 μM of BMS-345541 and 1, 10 μM of Purvalanol A. The stained gel below is a representative of the kinase reaction.
Mentions: We next focused our attention on BMS-345541 and asked whether this drug could inhibit the IKKβ kinase activity on its substrate IκBα. We immunoprecipitated (IP) IKKβ from both CEM (uninfected) and C8166 (infected) cells and used them in an in vitro kinase assays in the presence or absence of BMS-345541 (1.0 μM). Results are shown in Figure 1A where C8166 cells had far stronger IKKβ kinase activity as compared to CEM cells (compare lanes 2 to 4). Active kinases that were incubated with BMS-345541 showed a reduction of activity from both infected and uninfected cell extracts. However, the inhibition was much more dramatic with kinases isolated from HTLV-1 infected cells. We next titrated various levels of BMS-345541 for both kinases in our in vitro assay. Results are shown in Panel B where 0.01, 0.1, and 1.0 μM of BMS-345541 were used for a complete range of titrations. Interestingly, at 0.1 μM there was a significant reduction in the kinase activity from infected cells (lane 4 compared to lane 9). A control drug, Purvalanol A, which is a CDK inhibitor, did not inhibit the IKKβ kinase activity obtained from infected cells. Collectively, these results indicate that IKKβ from infected cells is much more sensitive to BMS-345541 as compared to IKKβ from uninfected cells.

Bottom Line: The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us.The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage.The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target both NF-kappaB and CDK complex and the G1/S border, might be promising new agents in the treatment of these infected patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, George Washington University School of Medicine, Washington, District of Columbia 20037, USA.

ABSTRACT
Human T-cell leukemia virus type-1 (HTLV-1) induces adult T-cell leukemia/lymphoma (ATL/L), a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-kappaB and the cell cycle pathways. The observation that NF-kappaB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-kappaB and CDK inhibitors (total of 35 compounds) to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKbeta kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration) value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target both NF-kappaB and CDK complex and the G1/S border, might be promising new agents in the treatment of these infected patients.

No MeSH data available.


Related in: MedlinePlus