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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 3. Reversibility of PXD101 responses. (A and B) DB (A) or OCI-Ly19 (B) cells were treated at their respective IC50 concentrations for 0, 2, 4, 8, or 24 h after which the cells were washed and resuspended in fresh medium without drug. After 24 h further incubation, the cells were harvested and subjected to Annexin V/PI assay. (C–F) SUDHL4 (C), U2932 (D), and SUDHL8 (E) were treated with either DMSO or PXD101 (at IC50 concentrations) for 48 h. Cells were then washed and incubated in drug-free medium for a further 0, 24, 48, or 72 h. The cells were harvested and subjected to cell cycle analysis. The graphs shown represent the results of 3–4 independent experiments. Error bars represent SEM.
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Figure 3: Figure 3. Reversibility of PXD101 responses. (A and B) DB (A) or OCI-Ly19 (B) cells were treated at their respective IC50 concentrations for 0, 2, 4, 8, or 24 h after which the cells were washed and resuspended in fresh medium without drug. After 24 h further incubation, the cells were harvested and subjected to Annexin V/PI assay. (C–F) SUDHL4 (C), U2932 (D), and SUDHL8 (E) were treated with either DMSO or PXD101 (at IC50 concentrations) for 48 h. Cells were then washed and incubated in drug-free medium for a further 0, 24, 48, or 72 h. The cells were harvested and subjected to cell cycle analysis. The graphs shown represent the results of 3–4 independent experiments. Error bars represent SEM.

Mentions: To investigate the relationship between the extent of drug exposure and the cellular response, we performed drug removal experiments. The cell lines which have a cytotoxic response to PXD101 were exposed to PXD101 for various times up to 24 h prior to drug removal. After 24 h further incubation in drug-free medium, they were examined for evidence of apoptosis (Fig. 3A and B). The results show that only 8–24 h exposure to PXD101 is sufficient to induce similar amounts of cell death in DB and OCI-Ly19 cells as observed with 48 h of continuous exposure (compare with Fig. 1A and C, 48 h). This indicates that the cells become committed to the apoptotic program within 24 h of treatment such that continued exposure to PXD101 is not required. Thus, we classify this group of cell lines as sensitive to HDACi.


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 3. Reversibility of PXD101 responses. (A and B) DB (A) or OCI-Ly19 (B) cells were treated at their respective IC50 concentrations for 0, 2, 4, 8, or 24 h after which the cells were washed and resuspended in fresh medium without drug. After 24 h further incubation, the cells were harvested and subjected to Annexin V/PI assay. (C–F) SUDHL4 (C), U2932 (D), and SUDHL8 (E) were treated with either DMSO or PXD101 (at IC50 concentrations) for 48 h. Cells were then washed and incubated in drug-free medium for a further 0, 24, 48, or 72 h. The cells were harvested and subjected to cell cycle analysis. The graphs shown represent the results of 3–4 independent experiments. Error bars represent SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926892&req=5

Figure 3: Figure 3. Reversibility of PXD101 responses. (A and B) DB (A) or OCI-Ly19 (B) cells were treated at their respective IC50 concentrations for 0, 2, 4, 8, or 24 h after which the cells were washed and resuspended in fresh medium without drug. After 24 h further incubation, the cells were harvested and subjected to Annexin V/PI assay. (C–F) SUDHL4 (C), U2932 (D), and SUDHL8 (E) were treated with either DMSO or PXD101 (at IC50 concentrations) for 48 h. Cells were then washed and incubated in drug-free medium for a further 0, 24, 48, or 72 h. The cells were harvested and subjected to cell cycle analysis. The graphs shown represent the results of 3–4 independent experiments. Error bars represent SEM.
Mentions: To investigate the relationship between the extent of drug exposure and the cellular response, we performed drug removal experiments. The cell lines which have a cytotoxic response to PXD101 were exposed to PXD101 for various times up to 24 h prior to drug removal. After 24 h further incubation in drug-free medium, they were examined for evidence of apoptosis (Fig. 3A and B). The results show that only 8–24 h exposure to PXD101 is sufficient to induce similar amounts of cell death in DB and OCI-Ly19 cells as observed with 48 h of continuous exposure (compare with Fig. 1A and C, 48 h). This indicates that the cells become committed to the apoptotic program within 24 h of treatment such that continued exposure to PXD101 is not required. Thus, we classify this group of cell lines as sensitive to HDACi.

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