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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 5. PXD101 treatment induces loss of Rb protein and Rb phosphorylation. (A and B) The cell lines shown were treated with PXD101 for up to 72 h. (A) Whole cell extracts were subjected to western blotting with antibodies against total Rb protein or α-tubulin. (B) Total RNA was extracted from cells and used to measure levels of Rb mRNA by RT-qPCR. (C and D) Whole cell extracts from PXD101-treated SUDHL4 (C) or SUDHL8 (D) cells were subjected to western blotting with antibodies against Rb phosphorylated at either Ser780 or Ser795, hypophosphorylated Rb, or α-tubulin. (E and F) Levels of total Rb, pRb Ser780, and pRb Ser795 were quantitated from non-saturated images and normalized to levels of α-tubulin for SUDHL4 (E) and SUDHL8 (F) cells. Normalized values from each timepoint of PXD101 treatment are expressed as fractions or multiples of the normalized value from untreated cells for each individual experiment. All of the results shown are representative of 2–4 independent experiments.
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Figure 5: Figure 5. PXD101 treatment induces loss of Rb protein and Rb phosphorylation. (A and B) The cell lines shown were treated with PXD101 for up to 72 h. (A) Whole cell extracts were subjected to western blotting with antibodies against total Rb protein or α-tubulin. (B) Total RNA was extracted from cells and used to measure levels of Rb mRNA by RT-qPCR. (C and D) Whole cell extracts from PXD101-treated SUDHL4 (C) or SUDHL8 (D) cells were subjected to western blotting with antibodies against Rb phosphorylated at either Ser780 or Ser795, hypophosphorylated Rb, or α-tubulin. (E and F) Levels of total Rb, pRb Ser780, and pRb Ser795 were quantitated from non-saturated images and normalized to levels of α-tubulin for SUDHL4 (E) and SUDHL8 (F) cells. Normalized values from each timepoint of PXD101 treatment are expressed as fractions or multiples of the normalized value from untreated cells for each individual experiment. All of the results shown are representative of 2–4 independent experiments.

Mentions: Progression through G1 is unhindered in the PXD101-sensitive cell lines but blocked in the resistant lines. Therefore, we examined the expression and phosphorylation of proteins that regulate G1 progression. Inactivation of the retinoblastoma protein (Rb) through phosphorylation is a key event that allows G1 progression through the restriction point, and HDACi have been shown to cause decreased Rb phosphorylation in some cell types.29-32 Since phosphorylation of Rb slows its migration through SDS-PAGE gels, we first used an antibody against total Rb to determine whether PXD101 changes Rb mobility. While we found that U2932 cells do not express detectable levels of Rb protein, Figure 5A shows that Rb mobility increases with the length of PXD101 treatment in all the other cell lines, indicating a shift from hyperphosphorylated to hypophosphorylated Rb. This shift was confirmed with the use of antibodies against the latter. Figure 5C and D show the accumulation of hypophosphorylated Rb in SUDHL4 and SUDHL8 cells. Surprisingly, PXD101 significantly downregulated total Rb levels in all Rb-expressing cell lines (Fig. 5A). In contrast, Rb mRNA is not significantly downregulated in any of the cell lines with the exception of OCI-Ly19, where it decreases by about 40%. In DB and SUDHL4 cells Rb mRNA levels are upregulated by PXD101. This contrasts with an 80% decrease in total Rb protein, as shown for SUDHL4 and SUDHL8 cells (Fig. 5E and F). These results indicate that Rb levels are regulated by post-transcriptional mechanisms in response to PXD101 treatment. It is noteworthy that U2932 cells have Rb mRNA but little to no Rb protein, suggesting that the cells have at least one intact and actively-transcribed copy of the Rb gene. In fact Rb mRNA levels in U2932 cells are not significantly different from those measured in SUDHL8 (Fig. S1B). Altogether these observations imply that DLBCL cells have robust post-transcriptional mechanisms to regulate expression of Rb protein.


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 5. PXD101 treatment induces loss of Rb protein and Rb phosphorylation. (A and B) The cell lines shown were treated with PXD101 for up to 72 h. (A) Whole cell extracts were subjected to western blotting with antibodies against total Rb protein or α-tubulin. (B) Total RNA was extracted from cells and used to measure levels of Rb mRNA by RT-qPCR. (C and D) Whole cell extracts from PXD101-treated SUDHL4 (C) or SUDHL8 (D) cells were subjected to western blotting with antibodies against Rb phosphorylated at either Ser780 or Ser795, hypophosphorylated Rb, or α-tubulin. (E and F) Levels of total Rb, pRb Ser780, and pRb Ser795 were quantitated from non-saturated images and normalized to levels of α-tubulin for SUDHL4 (E) and SUDHL8 (F) cells. Normalized values from each timepoint of PXD101 treatment are expressed as fractions or multiples of the normalized value from untreated cells for each individual experiment. All of the results shown are representative of 2–4 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Figure 5. PXD101 treatment induces loss of Rb protein and Rb phosphorylation. (A and B) The cell lines shown were treated with PXD101 for up to 72 h. (A) Whole cell extracts were subjected to western blotting with antibodies against total Rb protein or α-tubulin. (B) Total RNA was extracted from cells and used to measure levels of Rb mRNA by RT-qPCR. (C and D) Whole cell extracts from PXD101-treated SUDHL4 (C) or SUDHL8 (D) cells were subjected to western blotting with antibodies against Rb phosphorylated at either Ser780 or Ser795, hypophosphorylated Rb, or α-tubulin. (E and F) Levels of total Rb, pRb Ser780, and pRb Ser795 were quantitated from non-saturated images and normalized to levels of α-tubulin for SUDHL4 (E) and SUDHL8 (F) cells. Normalized values from each timepoint of PXD101 treatment are expressed as fractions or multiples of the normalized value from untreated cells for each individual experiment. All of the results shown are representative of 2–4 independent experiments.
Mentions: Progression through G1 is unhindered in the PXD101-sensitive cell lines but blocked in the resistant lines. Therefore, we examined the expression and phosphorylation of proteins that regulate G1 progression. Inactivation of the retinoblastoma protein (Rb) through phosphorylation is a key event that allows G1 progression through the restriction point, and HDACi have been shown to cause decreased Rb phosphorylation in some cell types.29-32 Since phosphorylation of Rb slows its migration through SDS-PAGE gels, we first used an antibody against total Rb to determine whether PXD101 changes Rb mobility. While we found that U2932 cells do not express detectable levels of Rb protein, Figure 5A shows that Rb mobility increases with the length of PXD101 treatment in all the other cell lines, indicating a shift from hyperphosphorylated to hypophosphorylated Rb. This shift was confirmed with the use of antibodies against the latter. Figure 5C and D show the accumulation of hypophosphorylated Rb in SUDHL4 and SUDHL8 cells. Surprisingly, PXD101 significantly downregulated total Rb levels in all Rb-expressing cell lines (Fig. 5A). In contrast, Rb mRNA is not significantly downregulated in any of the cell lines with the exception of OCI-Ly19, where it decreases by about 40%. In DB and SUDHL4 cells Rb mRNA levels are upregulated by PXD101. This contrasts with an 80% decrease in total Rb protein, as shown for SUDHL4 and SUDHL8 cells (Fig. 5E and F). These results indicate that Rb levels are regulated by post-transcriptional mechanisms in response to PXD101 treatment. It is noteworthy that U2932 cells have Rb mRNA but little to no Rb protein, suggesting that the cells have at least one intact and actively-transcribed copy of the Rb gene. In fact Rb mRNA levels in U2932 cells are not significantly different from those measured in SUDHL8 (Fig. S1B). Altogether these observations imply that DLBCL cells have robust post-transcriptional mechanisms to regulate expression of Rb protein.

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