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A model of sensitivity and resistance to histone deacetylase inhibitors in diffuse large B cell lymphoma: Role of cyclin-dependent kinase inhibitors.

Tula-Sanchez AA, Havas AP, Alonge PJ, Klein ME, Doctor SR, Pinkston W, Glinsmann-Gibson BJ, Rimsza LM, Smith CL - Cancer Biol. Ther. (2013)

Bottom Line: While the initial treatment strategy is highly effective, relapse occurs in 40% of cases.Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation.PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G 1 arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology; College of Pharmacy; University of Arizona; Tucson, AZ USA.

ABSTRACT
Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDACi monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDACi, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G 2/M arrest and apoptosis and resistance by reversible G 1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDACi might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDACi resistance. Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G 1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDACi against aggressive DLBCL.

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Figure 7. PXD101 effects on expression of cyclin-dependent kinase inhibitors (CKI) p21 and p27 and modification of p53. (A and B) The cell lines shown were treated with PXD101 for 0, 2, 4, 8, 24, 48, and 72 h. Whole cell extracts were generated and subjected to western blotting with antibodies against p21, p27, hsp90, α-tubulin, or GAPDH. PXD101-resistant cell lines are shown in (A) while PXD101-sensitive cell lines are shown in (B). (C and D) Cells were treated with PXD101 (P), doxorubicin (2 μM) (D), or Nutlin-3A (10 or 20 μM) (N) for 8 or 24 h. Whole cell extracts from treated and untreated cells were generated and subjected to western blotting with the antibodies indicated. PXD101-resistant cell lines are shown in (C) while PXD101-sensitive cell lines are shown in (D). The blots shown are representative of 3 independent experiments.
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Figure 7: Figure 7. PXD101 effects on expression of cyclin-dependent kinase inhibitors (CKI) p21 and p27 and modification of p53. (A and B) The cell lines shown were treated with PXD101 for 0, 2, 4, 8, 24, 48, and 72 h. Whole cell extracts were generated and subjected to western blotting with antibodies against p21, p27, hsp90, α-tubulin, or GAPDH. PXD101-resistant cell lines are shown in (A) while PXD101-sensitive cell lines are shown in (B). (C and D) Cells were treated with PXD101 (P), doxorubicin (2 μM) (D), or Nutlin-3A (10 or 20 μM) (N) for 8 or 24 h. Whole cell extracts from treated and untreated cells were generated and subjected to western blotting with the antibodies indicated. PXD101-resistant cell lines are shown in (C) while PXD101-sensitive cell lines are shown in (D). The blots shown are representative of 3 independent experiments.

Mentions: The activity of the cyclin E/cdk2 complex is inhibited by association with the CIP/KIP family of cyclin-dependent kinase inhibitors (CKI), including p21, p27, and p57.36 HDACi have been shown to upregulate the expression of p21 in a wide variety of cell types, and there are several reports of increased p57 expression.35,37 Therefore, we treated cells for up to 72 h and monitored levels of the CKI by western blot. Levels of p57 were unchanged in all of the cell lines (not shown), but p21 and p27 levels were increased by PXD101 (Fig. 7A and B). Interestingly, in the cell lines sensitive to PXD101 p21 was only transiently upregulated (Fig. 7B), peaking at 6–8 h treatment in OCI-Ly19 and at 24 h in DB before returning to baseline levels. Levels of p27 were unaffected by PXD101 in OCI-Ly19 cells while in DB cells p27 levels increased at 72 h treatment, after the onset of apoptosis (Fig. 1A). In contrast, p21 was upregulated in a sustained fashion in the PXD101-resistant cell lines (Fig. 7A). p21 levels were increased by 6 h in SUDHL4 and U2932 cells and elevated levels were maintained out to 72 h. SUDHL8 cells do not express detectable levels of p21. Levels of p27 increased significantly in all the PXD101-resistant cell lines by 24 h treatment.


A model of sensitivity and resistance to histone deacetylase inhibitors in diffuse large B cell lymphoma: Role of cyclin-dependent kinase inhibitors.

Tula-Sanchez AA, Havas AP, Alonge PJ, Klein ME, Doctor SR, Pinkston W, Glinsmann-Gibson BJ, Rimsza LM, Smith CL - Cancer Biol. Ther. (2013)

Figure 7. PXD101 effects on expression of cyclin-dependent kinase inhibitors (CKI) p21 and p27 and modification of p53. (A and B) The cell lines shown were treated with PXD101 for 0, 2, 4, 8, 24, 48, and 72 h. Whole cell extracts were generated and subjected to western blotting with antibodies against p21, p27, hsp90, α-tubulin, or GAPDH. PXD101-resistant cell lines are shown in (A) while PXD101-sensitive cell lines are shown in (B). (C and D) Cells were treated with PXD101 (P), doxorubicin (2 μM) (D), or Nutlin-3A (10 or 20 μM) (N) for 8 or 24 h. Whole cell extracts from treated and untreated cells were generated and subjected to western blotting with the antibodies indicated. PXD101-resistant cell lines are shown in (C) while PXD101-sensitive cell lines are shown in (D). The blots shown are representative of 3 independent experiments.
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Related In: Results  -  Collection

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Figure 7: Figure 7. PXD101 effects on expression of cyclin-dependent kinase inhibitors (CKI) p21 and p27 and modification of p53. (A and B) The cell lines shown were treated with PXD101 for 0, 2, 4, 8, 24, 48, and 72 h. Whole cell extracts were generated and subjected to western blotting with antibodies against p21, p27, hsp90, α-tubulin, or GAPDH. PXD101-resistant cell lines are shown in (A) while PXD101-sensitive cell lines are shown in (B). (C and D) Cells were treated with PXD101 (P), doxorubicin (2 μM) (D), or Nutlin-3A (10 or 20 μM) (N) for 8 or 24 h. Whole cell extracts from treated and untreated cells were generated and subjected to western blotting with the antibodies indicated. PXD101-resistant cell lines are shown in (C) while PXD101-sensitive cell lines are shown in (D). The blots shown are representative of 3 independent experiments.
Mentions: The activity of the cyclin E/cdk2 complex is inhibited by association with the CIP/KIP family of cyclin-dependent kinase inhibitors (CKI), including p21, p27, and p57.36 HDACi have been shown to upregulate the expression of p21 in a wide variety of cell types, and there are several reports of increased p57 expression.35,37 Therefore, we treated cells for up to 72 h and monitored levels of the CKI by western blot. Levels of p57 were unchanged in all of the cell lines (not shown), but p21 and p27 levels were increased by PXD101 (Fig. 7A and B). Interestingly, in the cell lines sensitive to PXD101 p21 was only transiently upregulated (Fig. 7B), peaking at 6–8 h treatment in OCI-Ly19 and at 24 h in DB before returning to baseline levels. Levels of p27 were unaffected by PXD101 in OCI-Ly19 cells while in DB cells p27 levels increased at 72 h treatment, after the onset of apoptosis (Fig. 1A). In contrast, p21 was upregulated in a sustained fashion in the PXD101-resistant cell lines (Fig. 7A). p21 levels were increased by 6 h in SUDHL4 and U2932 cells and elevated levels were maintained out to 72 h. SUDHL8 cells do not express detectable levels of p21. Levels of p27 increased significantly in all the PXD101-resistant cell lines by 24 h treatment.

Bottom Line: While the initial treatment strategy is highly effective, relapse occurs in 40% of cases.Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation.PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G 1 arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology; College of Pharmacy; University of Arizona; Tucson, AZ USA.

ABSTRACT
Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDACi monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDACi, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G 2/M arrest and apoptosis and resistance by reversible G 1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDACi might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDACi resistance. Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G 1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDACi against aggressive DLBCL.

Show MeSH
Related in: MedlinePlus