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Blood, Vol. 105, Issue 1, 308-316, January 1, 2005
The phosphodiesterase PDE4B limits cAMP-associated PI3K/AKT–dependent apoptosis in diffuse large B-cell lymphoma
Blood Smith et al.
105: 308
Supplemental materials for: Smith et al, Vol 105, Issue 1, 308-316
Cloning of the human PDE4B5 isoform. While characterizing the expression of PDE4B isoforms in normal B cells and DLBCL cell lines, we cloned a novel PDE4B transcriptional unit, which we termed PDE4B5. This novel sequence includes the upstream conserved regions 1 and 2 (UCR1 and UCR2) typical of the PDE4B long isoforms, PDE4B1 and PDE4B3. However, the PDE4B5 sequence differs from those of B1 and B3 upstream of the common long isoform region (Shepherd M, McSorley T, Olsen AE, et al. Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform. Biochem J. 2003;370:429-438). The PDE4B5 specific sequences include an in-frame, Kozak consensus, an initiation codon, and a distinct, short (6-amino-acid) N-terminus region. At the genomic level, the unique PDE4B5 sequences are closer to the common UCR1 than those of B3 and B1 (at 198, 254, and 328 kb, respectively), with the general organization of the 580-kb PDE4B locus being B1-B3-B5-B2, telomeric to centromeric (genomic contig accession number NT_032977). Note that PDE4B5 does not represent the human homolog of the recently described rat PDE4B4 isoform (Shepherd et al).
Files in this Data Supplement:
- Table S1. Activity profile of PLX513 against a set of PDE catalytic domains (PDF, 73 KB) -
The data are displayed as the 50% inhibitory concentration (IC50) values for PLX513 against the catalytic domains of PDE1B, PDE2A, PDE3B, PDE4B, PDE4D, PDE5A, PDE7B, PDE10A, and PDE11A. PLX513 is 10- to 100-fold more potent toward PDE4B than toward other PDEs, including members of the PDE4 family (4D), additional cAMP-specific PDEs (PDE7B), and B-cell relevant dual-specificity phosphodiesterases (PDE3B).
- Figure S1. Chromatogram of the human PDE4B5 sequence (PDF, 40 KB) -
The novel unique PDE4B5 sequence is indicated by the green bar; previously described common long isoform region sequences, by the red bar. The start codon (ATG) is underlined in blue, and the upstream stop codon (TGA) is indicated by the star.
- Figure S2. Overall survival curve by PDE4B2 expression for 112 patients with DLBCL (PDF, 120 KB) -
Kaplan-Meier estimates for overall survival, comparing the 56 patients with PDE4B2 below the median (low PDE4B) with the 56 patients with pde4b2 above the median (high PDE4B2) (P=.08, log rank test). At 5 years from diagnosis, the survival in the patients with low expression was 63%, compared with 47% for those with high expression. Median survival has not been reached for the low PDE4B group; median survival is 4.8 years for the high PDE4B group. Median follow-up for the surviving patients is 6.4 years (range, 2.3 to 12.2 years)
- Figure S3. Expression of PDE4B isoforms (PDF, 54 KB) -
(A) Western blot analysis of endogenous and retrovirally expressed FLAG-tagged PDE4B isoforms: PDE4B blot. Whole-cell lysates were resolved by 10% SDS-PAGE and immunoblotted with the PDE4B-specific antisera (see "Materials and methods"). Lymphoma cell lines expressing predominantly PDE4B3 (lane 1) and PDE4B2 (lane 2) and a PDE4B-low line transduced with either MSCV-GFP alone (lane 3) or MSCV-PDE4B1 (lane 4), -PDE4B2 (lane 5), or -PDE4B3 (lane 6) are shown. The arrows indicate the sizes of endogenous and transduced PDE4B isoforms. Note that the transduced PDE4B species are slightly larger than the endogenous PDE4B isoforms because of the added triple-FLAG sequences. (B) Western blot analysis of retrovirally expressed PDE4B isoforms: FLAG blot. As in panel A, lysates from cells expressing FLAG-tagged MSCV-GFP alone (lane 1), or MSCV-PDE4B1 (lane 2), -PDE4B2 (lane 3), or -PDE4B3 (lane 4) were resolved by 10% SDS-PAGE and immunoblotted with an anti-FLAG antibody. The PDE4B2 isoform doublet is depicted.
- Figure S4. cAMP-induced growth inhibition and apoptosis is independent of PKA (PDF, 47 KB) -
(A) Determination of PKA activity in DHL6 cells treated with forskolin/rolipram in the presence or absence of H-89. Cells were incubated with 40 µM forskolin and 10 µM rolipram for 30 minutes without pretreatment or following pretreatment with 10 µM H-89, the PKA inhibitor, for 30 minutes. (B) Proliferation of DHL6 cells treated with forskolin/rolipram in the presence or absence of H-89. Cells were treated with 40 µM forskolin and 10 µM rolipram for 24 hours, with or without pre-treatment with 10 µM H-89 for 30 minutes, or with H-89 alone for 24 hours. Data represent the mean and SD of 3 independent experiments.
- Figure S5. cAMP-induced growth inhibition and apoptosis is independent of PKA (PDF, 97 KB) -
(A) cAMP effects on RAP1 activation in DLBCL cell lines. DHL6 cells were starved overnight in 1% FBS media and incubated with 40 µM forskolin and 10 µM rolipram for 2 and 15 minutes or with 10µg/ml of goat anti–human IgM for 1 minute. OCI-Ly3 cells were serum-starved for 6 hours. Cells lysates were harvested, assayed for RAP1 activation, and immunoblotted with anti-RAP1 antisera. (B) Effects of EPAC-specific cAMP analogues on RAP1 activation in DLBCL cell lines and PC-12 cells. DHL6 and PC12 cells were serum starved overnight followed by addition of 0.5 mM 8-pCPT-2′-O-Me-cAMP or 40 µM forskolin and 10 µM rolipram for the indicated time periods. The EPAC-specific cAMP analogue did not increase RAP1 activation in DLBCL cells, although the analogue increased RAP1 activity in EPAC-responsive PC-12 cells. (C) Effects of EPAC-specific cAMP analogues on cellular proliferation. DHL6 cells were incubated with increasing concentrations of 0.001 to 1 mM 8-CPT-2'-O-Me-cAMP. Cell proliferation was determined by MTS assay. The EPAC-specific analogue did not inhibit the proliferation of the DLBCL cell line. (D) Semiquantitative analysis of EPAC1 expression in DLBCL cell lines. EPAC1 RT-PCR products were Southern blotted and hybridized with an internal oligonucleotide. Relative expression was determined by the densitometric ratio of band intensities between EPAC1 and a control gene (ABL). EPAC1 was expressed in the majority of the DLBCL cell lines analyzed, including DHL7 and OCI-Ly3, but was absent in DHL6.
- Figure S6. PP2A induction and effects on pAKT, pBAD, and cellular proliferation in DHL6 cells (PDF, 75 KB) -
(A) Effects of cAMP, okadaic acid, and H-89 on PP2A activity. DHL6 cells were either untreated or pretreated with 10 µM H89 for 30 minutes and incubated thereafter with 40 µM forskolin and 10 µM rolipram for 1 hour in the presence or absence of 5 nM okadaic acid (OA), the PP2A inhibitor, then PP2A activity was determined. Data shown are mean and SD of 3 independent experiments. In DHL6 cells, PP2A activity was significantly increased by forskolin/rolipram treatment. The PKA inhibitor, H-89, blocked PP2A induction, suggesting that the cAMP effects on PP2A activity are mediated via PKA. (B) Effects of PP2A activity on pAKT and pBAD levels. DHL6 cells were incubated with 40 µM forskolin and 10 µM rolipram, 5 nM okadaic acid alone, or all 3 compounds for 60 minutes. Thereafter, cell lysates were harvested and immunoblotted for pAKT (Ser473), pBAD (Ser136), or total AKT (loading control). (C) Effects of PP2A activity on cAMP-mediated inhibition of cell proliferation. DHL6 cells were incubated with 40 µM forskolin and 10 µM rolipram in the absence or presence of 5 nM okadaic acid for 24 hours, and cell proliferation was determined thereafter by MTS assay. The inhibition of PP2A with okadaic acid did not diminish the effects of cAMP on DHL6 cellular proliferation.
- Figure S7. PLX513 activity on PDE4B-high DLBCL cell lines (PDF, 142 KB) -
(A) Dose-dependency and specificity of PLX513’s effects on the proliferation of PDE4B-high DLBCL cell lines. OCI-Ly10 (left panel) and OCI-Ly3 (right panel) cells were treated with forskolin alone, forskolin and rolipram, forskolin and progressive doses of PLX513 (5-20 µM), or PLX513 alone for 24 hours. Data represent the ratio of proliferation of treated cells to cells cultured with vehicle alone for the same amounts of time. Cell proliferation was measured by MTS assay. Data represent the mean and SD of 3 independent experiments.
(B) cAMP levels are more efficiently increased following inhibition of PDE4B with PLX513 than with rolipram. OCI-Ly10 and OCI-Ly3 cells were incubated with 40 µM forskolin alone for 10 to 60 minutes or after preincubation with rolipram or PLX513 for 30 minutes. Results are displayed as the fold increase in cAMP levels in cells treated with forskolin alone versus those treated with forskolin and rolipram or forskolin and PLX513. Intracellular cAMP levels were determined by ELISA.
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