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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 induction of apoptosis in C8166. A) BMS-345541 induced caspase-3 and PARP cleavage C8166. MT-2, C8166, and CEM cells were treated with BMS-345541 at 0.1, 0.5, 1, and 5 μM for 48 hr. Total cell extracts were subjected to Western blot analysis for caspase-3 and PARP. β-actin Western blot was used as internal control. The results of caspase-3 were quantitated and normalized with β-actin. The ratio of c/un PARP was calculated by dividing cleaved PARP to un-cleaved PARP (data not shown). B) Detection of apoptosis through annexin V and PI staining. Cells were washed three times in PBS and re-suspended in binding buffer, stained with annexin V-FITC and PI for 15 minutes at room temperature. Analysis was performed on a BD FacsCalibur flow cytometer.
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Figure 2: BMS-345541 induction of apoptosis in C8166. A) BMS-345541 induced caspase-3 and PARP cleavage C8166. MT-2, C8166, and CEM cells were treated with BMS-345541 at 0.1, 0.5, 1, and 5 μM for 48 hr. Total cell extracts were subjected to Western blot analysis for caspase-3 and PARP. β-actin Western blot was used as internal control. The results of caspase-3 were quantitated and normalized with β-actin. The ratio of c/un PARP was calculated by dividing cleaved PARP to un-cleaved PARP (data not shown). B) Detection of apoptosis through annexin V and PI staining. Cells were washed three times in PBS and re-suspended in binding buffer, stained with annexin V-FITC and PI for 15 minutes at room temperature. Analysis was performed on a BD FacsCalibur flow cytometer.

Mentions: Resistance to cell apoptosis is one of the mechanisms that is important and is also required for the immortalization of T cells [63,66]. NF-κB signaling pathway is the survival pathway activated by HTLV-1 in order to keep the host cell active [67,68]. BMS-345541 targets IKKβ subunit which is responsible for activation of the NF-κB pathway [64,65]. To determine whether BMS-345541 can inhibit NF-κB pathway and induce apoptosis in HTLV-1 infected cells, we analyzed the level of apoptotic markers such as caspase-3 and PARP in both infected and uninfected cells. Caspase-3 is a member of cysteine protease and plays a key role in apoptosis [69]. When apoptosis is activated, the inactive pro-caspase-3 is processed into active large (17 kD) and small (12 kD) subunits [70]. PARP, poly(ADP-ribose) polymerase, is also an apoptosis marker that is cleaved from precursor form (116 kD) into active form (85 kD) by active caspase-3 during apoptosis [71-73]. Results in Figure 2A are Western blots that show titration of BMS-345541 in two infected and one uninfected cells. Samples were treated for 48 hours and extracts were made for Western blotting. The top panel shows the caspase Western and a gradual increase of p17 form in MT-2 cells as well as C8166 cells in concentrations between 0.5 and 1.0 μM. There was no change in the actin levels in any of the samples treated. Panel B shows the results of the Annexin V staining where live cells are represented at the bottom right corner box in each panel. All three samples were treated with 0.1 μM of BMS-345541 and stained for the presence of live and apoptotic cells. Interestingly both MT-2 and C8166 cells showed presence of few live cells as compared to CEM cells when treated with BMS-345541. Collectively, these data indicate that low concentrations of IKKβ inhibitor can apoptosis HTLV-1 cells much more efficiently as compared to 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 induction of apoptosis in C8166. A) BMS-345541 induced caspase-3 and PARP cleavage C8166. MT-2, C8166, and CEM cells were treated with BMS-345541 at 0.1, 0.5, 1, and 5 μM for 48 hr. Total cell extracts were subjected to Western blot analysis for caspase-3 and PARP. β-actin Western blot was used as internal control. The results of caspase-3 were quantitated and normalized with β-actin. The ratio of c/un PARP was calculated by dividing cleaved PARP to un-cleaved PARP (data not shown). B) Detection of apoptosis through annexin V and PI staining. Cells were washed three times in PBS and re-suspended in binding buffer, stained with annexin V-FITC and PI for 15 minutes at room temperature. Analysis was performed on a BD FacsCalibur flow cytometer.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: BMS-345541 induction of apoptosis in C8166. A) BMS-345541 induced caspase-3 and PARP cleavage C8166. MT-2, C8166, and CEM cells were treated with BMS-345541 at 0.1, 0.5, 1, and 5 μM for 48 hr. Total cell extracts were subjected to Western blot analysis for caspase-3 and PARP. β-actin Western blot was used as internal control. The results of caspase-3 were quantitated and normalized with β-actin. The ratio of c/un PARP was calculated by dividing cleaved PARP to un-cleaved PARP (data not shown). B) Detection of apoptosis through annexin V and PI staining. Cells were washed three times in PBS and re-suspended in binding buffer, stained with annexin V-FITC and PI for 15 minutes at room temperature. Analysis was performed on a BD FacsCalibur flow cytometer.
Mentions: Resistance to cell apoptosis is one of the mechanisms that is important and is also required for the immortalization of T cells [63,66]. NF-κB signaling pathway is the survival pathway activated by HTLV-1 in order to keep the host cell active [67,68]. BMS-345541 targets IKKβ subunit which is responsible for activation of the NF-κB pathway [64,65]. To determine whether BMS-345541 can inhibit NF-κB pathway and induce apoptosis in HTLV-1 infected cells, we analyzed the level of apoptotic markers such as caspase-3 and PARP in both infected and uninfected cells. Caspase-3 is a member of cysteine protease and plays a key role in apoptosis [69]. When apoptosis is activated, the inactive pro-caspase-3 is processed into active large (17 kD) and small (12 kD) subunits [70]. PARP, poly(ADP-ribose) polymerase, is also an apoptosis marker that is cleaved from precursor form (116 kD) into active form (85 kD) by active caspase-3 during apoptosis [71-73]. Results in Figure 2A are Western blots that show titration of BMS-345541 in two infected and one uninfected cells. Samples were treated for 48 hours and extracts were made for Western blotting. The top panel shows the caspase Western and a gradual increase of p17 form in MT-2 cells as well as C8166 cells in concentrations between 0.5 and 1.0 μM. There was no change in the actin levels in any of the samples treated. Panel B shows the results of the Annexin V staining where live cells are represented at the bottom right corner box in each panel. All three samples were treated with 0.1 μM of BMS-345541 and stained for the presence of live and apoptotic cells. Interestingly both MT-2 and C8166 cells showed presence of few live cells as compared to CEM cells when treated with BMS-345541. Collectively, these data indicate that low concentrations of IKKβ inhibitor can apoptosis HTLV-1 cells much more efficiently as compared to 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