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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

Purvalanol A induction of apoptosis in MT-2. A) Purvalanol A induced caspase-3 and PARP cleavage in MT-2 and C8166 cells. MT-2, C8166, and CEM were treated with Purvalanol A at various 0.1, 0.5, 1, and 5 μM for 48 hr. After 48 hr of treatment, total cell extracts were collected and subjected to Western blot analysis of caspase-3 and PARP. Westeron blot of β-actin was used as an internal control. B) Twenty five microgram of each Purvalanol A treated (5 μM for 48 hr) extracts from both MT-2 and C8166 cells were also used for western blot against cyclin A, E and actin.
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Figure 4: Purvalanol A induction of apoptosis in MT-2. A) Purvalanol A induced caspase-3 and PARP cleavage in MT-2 and C8166 cells. MT-2, C8166, and CEM were treated with Purvalanol A at various 0.1, 0.5, 1, and 5 μM for 48 hr. After 48 hr of treatment, total cell extracts were collected and subjected to Western blot analysis of caspase-3 and PARP. Westeron blot of β-actin was used as an internal control. B) Twenty five microgram of each Purvalanol A treated (5 μM for 48 hr) extracts from both MT-2 and C8166 cells were also used for western blot against cyclin A, E and actin.

Mentions: We have previously shown that cyclin E/CDK2 kinase activity is de-regulated in HTLV-1 infected cells and these cells are especially susceptible to Purvalanol A treatment [62]. Moreover, Purvalanol A, which is a purine analog that competes with the ATP binding site in CDKs, has been shown to inhibit cyclin E/CDK2 and cyclin A/CDK2 kinase activities with an IC50 of 0.035 and 0.07 μM, respectively [74-77]. We therefore treated both infected and uninfected cells for 48 hours with Purvalanol A and Western blotted for caspase-3 and PARP molecules. Results in Figure 4A show that the caspase-3 p17 molecule was present in infected cells treated with 0.1 and 0.5 μM of Purvalanol A (lanes 3 and 7). This was important since Purvalanol A did not significantly activate caspase-3 in CEM (lanes 11–15) or Jurkat cells (data not shown). There were no changes in actin (bottom panel), cyclin E, or cyclin A expression levels when treated with Purvalanol A (Panel B). Therefore Purvalanol A treatment induces caspase-3 activation in HTLV-1 infected, and not in uninfected cells. This is consistent with our previous results where Purvalanol A treatment of infected cells inhibited cyclin E/CDK2 complex activity in HTLV-1 infected cells, inhibited transcription of the LTR promoter and promoted apoptosis [62]. Along these lines, we also assayed for changes in cell cycle progression and apoptosis in these cells using FACS analysis. Results in Figure 5 show the titration of Purvalanol A for all three cell types. Interestingly, significant apoptosis appeared in infected cells treated at 1.0 and 5.0 μM concentrations.


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)

Purvalanol A induction of apoptosis in MT-2. A) Purvalanol A induced caspase-3 and PARP cleavage in MT-2 and C8166 cells. MT-2, C8166, and CEM were treated with Purvalanol A at various 0.1, 0.5, 1, and 5 μM for 48 hr. After 48 hr of treatment, total cell extracts were collected and subjected to Western blot analysis of caspase-3 and PARP. Westeron blot of β-actin was used as an internal control. B) Twenty five microgram of each Purvalanol A treated (5 μM for 48 hr) extracts from both MT-2 and C8166 cells were also used for western blot against cyclin A, E and actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Purvalanol A induction of apoptosis in MT-2. A) Purvalanol A induced caspase-3 and PARP cleavage in MT-2 and C8166 cells. MT-2, C8166, and CEM were treated with Purvalanol A at various 0.1, 0.5, 1, and 5 μM for 48 hr. After 48 hr of treatment, total cell extracts were collected and subjected to Western blot analysis of caspase-3 and PARP. Westeron blot of β-actin was used as an internal control. B) Twenty five microgram of each Purvalanol A treated (5 μM for 48 hr) extracts from both MT-2 and C8166 cells were also used for western blot against cyclin A, E and actin.
Mentions: We have previously shown that cyclin E/CDK2 kinase activity is de-regulated in HTLV-1 infected cells and these cells are especially susceptible to Purvalanol A treatment [62]. Moreover, Purvalanol A, which is a purine analog that competes with the ATP binding site in CDKs, has been shown to inhibit cyclin E/CDK2 and cyclin A/CDK2 kinase activities with an IC50 of 0.035 and 0.07 μM, respectively [74-77]. We therefore treated both infected and uninfected cells for 48 hours with Purvalanol A and Western blotted for caspase-3 and PARP molecules. Results in Figure 4A show that the caspase-3 p17 molecule was present in infected cells treated with 0.1 and 0.5 μM of Purvalanol A (lanes 3 and 7). This was important since Purvalanol A did not significantly activate caspase-3 in CEM (lanes 11–15) or Jurkat cells (data not shown). There were no changes in actin (bottom panel), cyclin E, or cyclin A expression levels when treated with Purvalanol A (Panel B). Therefore Purvalanol A treatment induces caspase-3 activation in HTLV-1 infected, and not in uninfected cells. This is consistent with our previous results where Purvalanol A treatment of infected cells inhibited cyclin E/CDK2 complex activity in HTLV-1 infected cells, inhibited transcription of the LTR promoter and promoted apoptosis [62]. Along these lines, we also assayed for changes in cell cycle progression and apoptosis in these cells using FACS analysis. Results in Figure 5 show the titration of Purvalanol A for all three cell types. Interestingly, significant apoptosis appeared in infected cells treated at 1.0 and 5.0 μM concentrations.

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