PDE7A is expressed in human B-lymphocytes and is up-regulated by elevation of intracellular cAMP
Introduction
Elevated intracellular levels of 3′,5′-cyclic adenosine monophosphate (cAMP) can induce apoptosis in susceptible subpopulations of both B- and T-lineage lymphocytes. This suggests that agents capable of modulating cAMP levels might be useful for the treatment of lymphoid malignancies [1]. One means of augmenting cAMP signaling has been through the use of cAMP phosphodiesterase (PDE) inhibitors, as inhibition of cAMP catabolism results in elevation of intracellular lymphoid cAMP levels in vivo [2]. Theophylline, a nonspecific methylxanthine PDE inhibitor, has been shown to induce apoptosis in chronic lymphocytic leukemia (CLL) B-lymphocytes in vitro [3], [4]. A subsequent Phase 2 clinical trial demonstrated that combined treatment with theophylline and chlorambucil induced positive responses in CLL patients who failed treatment with chlorambucil alone [5]. More recently, Makower et al. [6] have reported responses to theophylline monotherapy in three patients with advanced CLL.
Theophylline is a nonselective PDE inhibitor as well as an adenosine receptor antagonist, complicating both the clinical and research applications of this reagent. A more selective PDE inhibitor might also induce apoptosis in lymphoid cells and have therapeutic value in the treatment of lymphoid malignancies. Lymphoid cells contain several classes of cyclic nucleotide PDEs, including cGMP-inhibited PDE3 [7] and cAMP-specific PDE4 [8]. Inhibition of PDE4 with rolipram, an inhibitor specific for this class of enzymes, induces apoptosis of lymphocytes isolated from CLL patients [9]. Peripheral T-cells are resistant to rolipram-induced apoptosis and the susceptibility of CLL cells and T-cells to PDE4 inhibitors correlated with their sensitivity to apoptosis induced by the cell-permeable cAMP analog dibutyryl cAMP (dbcAMP) [9].
Since the initiation of our work on PDEs and CLL, interest has grown in the potential role of a novel cAMP PDE, PDE7A, in lymphoid cyclic nucleotide metabolism. PDE7A was originally cloned by Michaeli et al. [10] as a result of its ability to complement the heat-shock-sensitive phenotype of a Saccharomyces cerevisiae strain in which two yeast cAMP PDE genes had been disrupted. There are two splice variants of the PDE7A gene, encoding enzymes with differing sequence and tissue distribution. PDE7A1, which contains a unique hydrophilic 45 residue NH2-terminus sequence, has been detected in lymphoid cells and migrates with an apparent MW of 55 kDa [11]. In contrast, PDE7A2, which contains a unique hydrophobic 20 residue N-terminus sequence, was cloned from skeletal muscle cDNA libraries and migrates with an apparent MW of 50 kDa [12]. A second member of the PDE7 gene family, PDE7B, was recently cloned [13], [14], [15]. Expression of PDE7B was detected in multiple tissues, but not in lymphoid cells.
PDE7A1 has been detected in both primary T-cells and cell lines derived from T-cells [11], [16]. PDE7A is reportedly up-regulated in T-cells upon costimulation with CD3 and CD28 and is required for T-cell proliferation [17]. The expression of PDE7A in B-lymphocytes is not as well documented. Gantner et al. [18] reported substantial PDE activity in primary human B-cells that were not inhibited by inhibitors specific for PDE1, PDE3, or PDE4. This residual cAMP-hydrolyzing activity was attributed to PDE7. Li et al. [17] examined the expression of PDE7A in two human B-cell lines, Jijoye and Ramos. While PDE7A transcript was detected in the B-cell lines, no PDE7A protein was detected in these cells by Western analysis.
The goal of the current study was to determine whether PDE7A is expressed in CLL B-lymphocytes and to assess whether the activity of this enzyme can be inhibited by methylxanthines. In this report, we demonstrate that PDE7A is expressed in normal B-cells, primary CLL cells, and the CLL B-cell line WSU-CLL. Furthermore, we demonstrate that PDE7A protein expression is up-regulated in WSU-CLL cells in response to treatment with theophylline and IBMX, suggesting a feedback mechanism that enables additional control of cellular cAMP levels. Lastly, we demonstrate similar changes in PDE7 protein expression in response to treatment with a selective PDE7A inhibitor.
Section snippets
Reagents
DPCPX and rolipram were from RBI (Natick, MA). Forskolin and IBMX were from Sigma (St. Louis, MO). Theophylline was obtained as a 3.2-mg/ml solution in 5% dextrose (Baxter Healthcare, Deerfield, IL). H-89 was from Calbiochem (San Diego, CA). Compounds IC242 and IC243 were obtained from ICOS, Bothell, WA.
Cell purification and culture
WSU-CLL cells were generously provided by Dr. R. Mohamed (Wayne State University). Primary leukemic cells were isolated from the peripheral blood of patients with acute T-cell leukemia (ATL) or
Results
We initially sought to determine whether PDE7A is expressed in human lymphoid cells from patients with CLL. Western analysis was used to assess the expression of PDE7A in isolated splenic B-cells, T-cells, and lymphoid cells from patients with either ATL or CLL (Fig. 1A). PDE7A was readily detected in isolated primary splenic B-cells as well as primary peripheral blood T-cells isolated from healthy individuals. In addition, PDE7A was readily detected in lymphoid cells from a CLL patient. In
Discussion
We find constitutive expression of PDE7A in human splenic B-cells, peripheral blood T-cells, and in lymphoid cells from patients with CLL. In a CLL cell line, WSU-CLL, PDE7A expression is up-regulated by PDE inhibitors, such as theophylline and IBMX, as well as adenylate cyclase activators such as forskolin and the cAMP analogue, dbcAMP. As each of these stimuli raise cAMP levels in WSU-CLL cells (Fig. 7 and data not shown), this would suggest that the observed up-regulation of PDE7A is a
Acknowledgements
We thank the following ICOS scientists for their assistance in this project: Dr. Kate Loughney, Dr. Timothy Martins, and Guy Rosman for helpful discussions regarding PDE7 and methylxanthines; Cathy Farrell and Janelle Taylor for help with antibody development; and Dr. Vince Florio and Lothar Uher for help with the PDE assays.
This work is supported by grants from the Leukemia Society of America, the American Society of Clinical Oncology, and the National Cancer Institute (RO1 CA79838-01A2).
References (28)
- et al.
Gen Pharmacol
(1998) - et al.
Blood
(1996) - et al.
Blood
(1998) - et al.
J Biol Chem
(1993) - et al.
J Biol Chem
(1997) - et al.
Biochem Biophys Res Commun
(2000) - et al.
Biochem Biophys Res Commun
(2000) - et al.
Cell Signal
(1997) - et al.
Genomics
(1996) - et al.
Life Sci
(1987)