Figure 1.
Trichostatin A and vorinostat induce PARP cleavage in a subset of DLBCL cell lines.
The indicated DLBCL cell lines were treated with either 300 nM TSA (top panel) or 3 µM vorinostat (bottom panel) for 24 h and Western blotting was performed for PARP and β-tubulin (as a normalizing control). Note: in cases where cells undergo extensive PARP cleavage (e.g., SUDHL4, Pfeiffer, SUDHL2), tubulin can appear under-loaded (likely because tubulin also begins to be degraded in these dying cells). Cell lines used were GCB-like (BJAB, SUDHL4, SUDHL6, Pfeiffer, Farage, and SUDHL8) and ABC-like (RC-K8 and SUDHL2). Grey font indicates HDACi-sensitive cell lines; black font indicates HDACi-resistant cell line SUDHL6. The arrowhead indicates cleaved PARP.
Figure 2.
Trichostatin A and vorinostat induce apoptosis and inhibit growth in a time- and dose-dependent manner.
(A) SUDHL4 and SUDHL6 cells were treated with either 300 nM TSA or 3 µM vorinostat for the indicated times. Whole-cell extracts were made, and Western blotting for PARP and β-tubulin (as a normalizing control) was performed. (B) SUDHL4 and SUDHL6 cells were treated for 14 h with the indicated concentrations of either TSA or vorinostat. PARP cleavage and β-tubulin levels (as a normalizing control) were assessed by Western blotting of whole-cell extracts. (C) Relative caspase-3 activity was measured in cells treated for 14 h with either TSA or vorinostat as indicated. The values represent the average relative fluorescence of three independent assays as compared to untreated samples (1.0). Error bars indicate standard error. (D) SUDHL4 and SUDHL6 cells were treated with 3 µM vorinostat for 14 h, cells were stained with acridine orange and ethidium bromide, and counted for live (black bars), necrotic (grey bars), and apoptotic (white bars) cells (see Materials and Methods). Each value is the average of three independent experiments and error bars indicate standard error. (E) Growth inhibition by vorinostat was assessed by treating SUDHL4 and SUDHL6 cells with increasing concentrations of vorinostat for 72 h, and cells were then counted using a hemocytometer. The relative numbers of cells are percentages as compared to cells grown for the same amount of time in the absence of vorinostat. The results are the averages of three separate treatment samples. Error bars indicate standard deviation. (F) SUDHL6 cells were treated with the indicated concentrations of vorinostat for 4 h. BIM and β-tubulin (as a normalizing control) were detected by Western blotting of whole-cell extracts. (G) SUDHL6 cells were treated for 24 h with the indicated concentrations of staurosporine. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts.
Figure 3.
HDACi sensitivity is decreased by over-expression of BCL-2 or BCL-XL and increased by over-expression of BIM.
Creation of four independent cell lines (SUDHL2 cells over-expressing BCL-2, SUDHL4 cells over-expressing BCL-XL, Farage cells over-expressing BCL-XL, and Farage cells over-expressing BIM) was confirmed by Western blotting for BCL-2, BCL-XL, and BIM (A–D) and compared to empty vector (–) transduced cells. β-tubulin was used as a normalizing control. (A) SUDHL2-BCL-2, (B) SUDHL4-BCL-XL, (C) Farage-BCL-XL, and (D) Farage-BIM cell lines were treated with either 300 nM TSA or 3 µM vorinostat for 24 h. Whole-cell extracts were made and Western blotting for PARP cleavage and β-tubulin (as a normalizing control) was performed. All cells over-expressing the protein of interest were compared to empty vector (–) transduced control cells.
Figure 4.
Vorinostat and the BH3 mimetic ABT-737 cooperate to increase apoptosis in DLBCL cell lines.
(A) SUDHL4, (B) Farage, (C) BJAB, (D) SUDHL6, and (E) RC-K8 cells were treated with ABT-737 in a dose-dependent manner with and without a sub-optimal level (0.5 µM) or a higher concentration (3 µM) of vorinostat. Whole-cell extracts were subjected to Western blotting to assess PARP cleavage and β-tubulin levels (as a normalizing control).
Figure 5.
SUDHL4 cells selected for vorinostat resistance show decreased sensitivity to vorinostat-induced apoptosis and cell proliferation inhibition.
(A) SUDHL4 and SUDHL4-VR cells were treated for 24 h with the indicated concentrations of vorinostat. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts. (B) Relative caspase-3 activity was measured in cells treated for 14 h with the indicated concentrations of vorinostat. The values represent the average relative fluorescence as compared to untreated samples (1.0) from three independent assays. Error bars indicate standard error. (C) Growth inhibition by vorinostat was assessed by treating SUDHL4-VR cells with increasing amounts of vorinostat for 72 h, and then counting cells using a hemocytometer. The relative numbers of cells are percentages as compared to cells incubated for the same amount of time in the absence of vorinostat. The results are the averages of three separate treatment samples. Error bars indicate standard deviation. (D) SUDHL4-VR cells were treated with the indicated concentrations of ABT-737 with and without a sub-optimal level (0.5 µM) of vorinostat. Whole-cell extracts were subjected to Western blotting for PARP cleavage and β-tubulin levels (as a normalizing control). (E) Relative caspase-3 activity was measured in cells treated for 14 h with TSA as indicated. The values represent the average relative fluorescence of three independent assays as compared to untreated samples. Error bars indicate standard error. (F) Cells were treated for 24 h with the indicated concentrations of staurosporine. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts. (G) Western blotting for the indicated BCL-2 family members in parental SUDHL4 cells and in two independent populations of SUDHL4-VR cells (1 and 2). β-tubulin was used as a protein loading control. The SUDHL4-VR(1) cell line was used for experiments in panels A–F.