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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 4. Sensitivity or resistance to PXD101 is not correlated with expression of MYC or BCL2. (A–C) Whole cell extracts were generated from the cell lines shown in 2–3 independent experiments. Equal amounts of protein were separated by SDS-PAGE and subjected to western transfer and immunoblotting with c-myc, BCL2, or GAPDH antibodies. All of the samples shown were run on the same SDS-PAGE gel and blotted simultaneously to accurately measure relative levels of each protein. The results of analysis are shown graphically for c-myc (B) and BCL2 (C). Levels of each protein are expressed relative to those in Exp. One of OCI-Ly19 cells. (D) Cells were treated with PXD101 for the times shown. Whole cell extracts were generated and equal amounts of protein were separated by SDS-PAGE, and subjected to Western transfer and immunoblotting with MYC, BCL2, or GAPDH antibodies. BCL2 shown for SUDHL8 correspond to the species with altered mobility as shown in (A). The results shown are representative of three independent experiments.
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Figure 4: Figure 4. Sensitivity or resistance to PXD101 is not correlated with expression of MYC or BCL2. (A–C) Whole cell extracts were generated from the cell lines shown in 2–3 independent experiments. Equal amounts of protein were separated by SDS-PAGE and subjected to western transfer and immunoblotting with c-myc, BCL2, or GAPDH antibodies. All of the samples shown were run on the same SDS-PAGE gel and blotted simultaneously to accurately measure relative levels of each protein. The results of analysis are shown graphically for c-myc (B) and BCL2 (C). Levels of each protein are expressed relative to those in Exp. One of OCI-Ly19 cells. (D) Cells were treated with PXD101 for the times shown. Whole cell extracts were generated and equal amounts of protein were separated by SDS-PAGE, and subjected to Western transfer and immunoblotting with MYC, BCL2, or GAPDH antibodies. BCL2 shown for SUDHL8 correspond to the species with altered mobility as shown in (A). The results shown are representative of three independent experiments.

Mentions: Expression of both MYC and BCL2 in DLBCL tumors has been associated with poor prognosis.5 Thus, we asked whether the MYC and BCL2 proteins are co-expressed in any of the DLBCL cell lines tested above. Equal amounts of multiple, independently-generated whole cell extracts from each cell line were run on an SDS-PAGE gel and subjected to immunoblotting with antibodies against MYC, BCL2, and GAPDH. Figure 4A shows that all cell lines tested co-express MYC and BCL2. We then considered whether overall levels of MYC or BCL2 in the various cell lines correlated with sensitivity or resistance to PXD101 because overexpression of BCL2 in Eμ-myc tumors in mice caused them to arrest in G1 in response to vorinostat treatment.22 The graphs in Figure 4B–D show levels of each protein in the various extracts expressed relative to levels of the same protein in one of the OCI-Ly19 extracts. In contrast to relatively even expression of GAPDH (Fig. 4D), the cell lines express varying levels of MYC and BCL2 (Fig. 4B and C). However, there is no clear relationship between relative levels of these two proteins and the cellular response to HDACi. In the case of MYC, the relative expression level was very similar in DB (PXD101-sensitive), SUDHL4, and U2932 (both PXD101-resistant) (Fig. 4A and B). For BCL2, levels in the PXD101-resistant cell lines were highly variable with SUDHL4 having the lowest levels of all 5 cell lines tested and U2932 having the highest (Fig. 4A and C). Interestingly, BCL2 from SUDHL8 cells reproducibly had a slower mobility in SDS-PAGE gels (Fig. 4A). It has been shown that phosphorylation or mutation of BCL2 can slow its mobility23 and mutations in BCL2 occur in 10–37% of GCB-type DLBCL tumors.24,25 Of all the cell lines shown, SUDHL8 had the lowest levels of BCL2 mRNA by far (Fig. S1A), consistent with the fact that it does not have the t(14;18) translocation that deregulates BCL2 transcription. Thus, because BCL2 protein levels in SUDHL8 cells are similar to those observed in other cell lines, its expression is likely determined by post-transcriptional mechanisms.


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 4. Sensitivity or resistance to PXD101 is not correlated with expression of MYC or BCL2. (A–C) Whole cell extracts were generated from the cell lines shown in 2–3 independent experiments. Equal amounts of protein were separated by SDS-PAGE and subjected to western transfer and immunoblotting with c-myc, BCL2, or GAPDH antibodies. All of the samples shown were run on the same SDS-PAGE gel and blotted simultaneously to accurately measure relative levels of each protein. The results of analysis are shown graphically for c-myc (B) and BCL2 (C). Levels of each protein are expressed relative to those in Exp. One of OCI-Ly19 cells. (D) Cells were treated with PXD101 for the times shown. Whole cell extracts were generated and equal amounts of protein were separated by SDS-PAGE, and subjected to Western transfer and immunoblotting with MYC, BCL2, or GAPDH antibodies. BCL2 shown for SUDHL8 correspond to the species with altered mobility as shown in (A). The results shown are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Figure 4. Sensitivity or resistance to PXD101 is not correlated with expression of MYC or BCL2. (A–C) Whole cell extracts were generated from the cell lines shown in 2–3 independent experiments. Equal amounts of protein were separated by SDS-PAGE and subjected to western transfer and immunoblotting with c-myc, BCL2, or GAPDH antibodies. All of the samples shown were run on the same SDS-PAGE gel and blotted simultaneously to accurately measure relative levels of each protein. The results of analysis are shown graphically for c-myc (B) and BCL2 (C). Levels of each protein are expressed relative to those in Exp. One of OCI-Ly19 cells. (D) Cells were treated with PXD101 for the times shown. Whole cell extracts were generated and equal amounts of protein were separated by SDS-PAGE, and subjected to Western transfer and immunoblotting with MYC, BCL2, or GAPDH antibodies. BCL2 shown for SUDHL8 correspond to the species with altered mobility as shown in (A). The results shown are representative of three independent experiments.
Mentions: Expression of both MYC and BCL2 in DLBCL tumors has been associated with poor prognosis.5 Thus, we asked whether the MYC and BCL2 proteins are co-expressed in any of the DLBCL cell lines tested above. Equal amounts of multiple, independently-generated whole cell extracts from each cell line were run on an SDS-PAGE gel and subjected to immunoblotting with antibodies against MYC, BCL2, and GAPDH. Figure 4A shows that all cell lines tested co-express MYC and BCL2. We then considered whether overall levels of MYC or BCL2 in the various cell lines correlated with sensitivity or resistance to PXD101 because overexpression of BCL2 in Eμ-myc tumors in mice caused them to arrest in G1 in response to vorinostat treatment.22 The graphs in Figure 4B–D show levels of each protein in the various extracts expressed relative to levels of the same protein in one of the OCI-Ly19 extracts. In contrast to relatively even expression of GAPDH (Fig. 4D), the cell lines express varying levels of MYC and BCL2 (Fig. 4B and C). However, there is no clear relationship between relative levels of these two proteins and the cellular response to HDACi. In the case of MYC, the relative expression level was very similar in DB (PXD101-sensitive), SUDHL4, and U2932 (both PXD101-resistant) (Fig. 4A and B). For BCL2, levels in the PXD101-resistant cell lines were highly variable with SUDHL4 having the lowest levels of all 5 cell lines tested and U2932 having the highest (Fig. 4A and C). Interestingly, BCL2 from SUDHL8 cells reproducibly had a slower mobility in SDS-PAGE gels (Fig. 4A). It has been shown that phosphorylation or mutation of BCL2 can slow its mobility23 and mutations in BCL2 occur in 10–37% of GCB-type DLBCL tumors.24,25 Of all the cell lines shown, SUDHL8 had the lowest levels of BCL2 mRNA by far (Fig. S1A), consistent with the fact that it does not have the t(14;18) translocation that deregulates BCL2 transcription. Thus, because BCL2 protein levels in SUDHL8 cells are similar to those observed in other cell lines, its expression is likely determined by post-transcriptional mechanisms.

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